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Welcome back to our “Thursday's Technique”-series, where this week, we are diving into the fascinating world of RNA modifications and their exploration through next-generation sequencing. We are about to uncover how bioinformatics and data analysis are pivotal in decoding the mysteries behind RNA modifications, such as m6A-seq. m6A-seq illuminates the nuanced yet impactful methylation at the N6-adenosine on RNA, profoundly influencing gene expression patterns and the progression of various diseases. Join us as we explore this cutting-edge method enabling scientists to map m6A modifications and understand their impact on biological processes, all through the lens of Next-Generation Sequencing!

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RNA modifications play a pivotal role in a variety of processes in cells such as gene expression regulation or RNA stability. m6A is the most abundant mRNA modification in mammalian cells (Ge et al, Nat Protoc 2023). m6A-seq enables researchers to effectively localise m6A modifications throughout the whole transcriptome and can be used for a broad range of applications.&nbsp;

What role do RNA modifications play in basic and translational research and how can they be examined?

RNA modifications play a critical role in modulating key cellular processes by regulating RNA metabolism and function. These modifications influence gene expression, RNA stability, and protein synthesis. While cells show an impressive range of more than 100 different possible RNA modifications for every existing type of RNA such as ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA) or long non-coding RNA (lncRNA), in this blogpost we mainly focus on the general Next-Generation Sequencing approaches that can be used to determine position and character of those modifications, especially focusing on N6-methyladenosine modification and sequencing of the same (m6A-seq). This is the most abundant RNA modification in mammalian cells. Details about specific modifications can be further researched in the sources we list below (Delaunay et al, Nat Rev Genet 2024).&nbsp;&nbsp;

The study of RNA modifications has been propelled by advancements in Next-Generation Sequencing technologies, allowing for transcriptome-wide mapping of RNA modifications. This has led to a deeper understanding of how RNA modifications can influence cell function under normal and disease conditions, offering new prospects for therapeutic intervention.&nbsp;&nbsp;

Pros + Comprehensive Detection: m6A-seq allows for transcriptome-wide identification of m6A modifications, providing insights into RNA methylation landscapes.&nbsp; + High Sensitivity and Specificity: It offers high sensitivity in detecting m6A sites, enabling the study of dynamic changes in RNA methylation.&nbsp; + Functional Insights: m6A-seq helps in understanding the biological significance of m6A modifications in gene expression regulation, RNA stability, and cellular processes.&nbsp;

Cons - Resolution Limits: While providing valuable information, m6A-seq does not achieve single-nucleotide resolution, limiting precise localisation of modifications.&nbsp; - Loss of Stoichiometry Information: The method focuses on enrichment and detection of methylated fragments, which can obscure the quantification of methylation levels across different RNA molecules.&nbsp; - Technical Complexity: The technique requires sophisticated bioinformatics tools for data analysis, posing challenges in interpretation and reproducibility.&nbsp;

- MeRIP-seq (Methylated RNA Immunoprecipitation Sequencing): Like m6A-seq, MeRIP-seq also enriches methylated RNA fragments before sequencing.&nbsp; - miCLIP (m6A Individual-nucleotide-resolution Crosslinking and Immunoprecipitation): Offers higher resolution than m6A-seq, pinpointing the exact location of m6A modifications.&nbsp;&nbsp; - PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation): Used for studying protein-RNA interactions but can be adapted to study specific RNA modifications by using antibodies against modified RNA.&nbsp;

How can RNA modifications be investigated via sequencing?

There are four approaches for sequencing RNA modifications: Direct sequencing, chemically assisted sequencing, antibody-based sequencing, and enzyme-assisted sequencing (Nanopore sequencing).&nbsp; Direct sequencing of RNA modifications utilises the fact that during reverse transcription PCR, the reverse transcriptase “stumbles” over modified RNA bases and therefore either aborts the synthesis or increasingly introduces mutation at these sites.&nbsp;&nbsp; For chemically assisted sequencing, chemical modifications are introduced to the RNA. Modified bases are protected from being changed or chemically modified and stay the same, which can then be detected via sequencing.&nbsp;&nbsp; Antibody-based sequencing methods are only available for a limited number of RNA modifications. Either the modification itself or the modifying enzymes can be targeted. For Nanopore sequencing, RNA does not have to be translated into cDNA, which makes it possible to conduct the sequencing without losing the actual modification. Single stranded RNA is guided through a small protein pore, which induces changes in the current inside the pore which are measured. Different bases, base combinations and RNA modifications modulate the current which can be translated into a sequence and give insight into modifications of the sequenced RNAs.&nbsp;

How can m6A-modifications be investigated via sequencing?

The N6-methyladenosine does not have an influence on base pairing properties or the introduction of chemical modifications (Dominissini et al, Nat Protoc 2013). Direct sequencing and chemically assisted sequencing will therefore not work in this case. A protocol presented by Dominissini et al (2013) proposes immunoprecipitation via a highly specific antibody targeting N6-methylated adenosines followed by sequencing.&nbsp;&nbsp; For immunoprecipitation of N6-methyladenosine sites followed by sequencing, the first steps include RNA isolation and careful fragmentation. Then, RNA-pulldown with an antibody that specifically binds to N6-methyladenosine sites is conducted leading to a sample containing only those RNA fragments that contain a N6-methyladenosine site. For creating a library suitable for sequencing, the enriched RNA fragments are amplified via PCR using random primers. After this first amplification, a second PCR must be conducted to add primers specific to the sequencing device you are going to use later. Now all that left is sequencing of your library and subsequent analysis of the data.&nbsp;

Applications of Next-Generation Sequencing for investigating RNA modifications

m6A is the most prevalent modification found in mammalian mRNA and plays a pivotal role in the nuanced regulation of gene expression. Through m6A sequencing, you can uncover the extensive impact of this modification across various biological fields, from understanding fundamental cellular processes to exploring its implications in disease mechanisms and potential therapeutic targets. The method opens a window into the molecular intricacies of RNA function, offering a broad spectrum of applications in both basic research and clinical studies:&nbsp;

m6A modifications play a role in RNA metabolism and gene regulation. For basic research it enables the precise mapping and quantification of m6A -modified sites across the transcriptome, uncovering its fundamental influence in cellular processes and disease mechanisms. In this research, Li et al (2023) used m6A -seq alongside RNA immunoprecipitation sequencing (RIP-seq) and transcriptomic co-analysis to investigate the molecular mechanisms underlying vascular system remodelling in response to acute hypoxia.&nbsp;

In neuroscience, m6A modifications play a critical role by influencing RNA stability and translation efficiency, thereby affecting gene expression patterns that are essential for brain development, synaptic plasticity, and the response to neural injury. Chen et al (2019) used m6A -seq to identify m6A -modified transcripts that are significantly involved in neurogenesis and neuronal development, revealing the pivotal role of m6A modification in regulating gene expression critical for neural stem cell differentiation and neuronal maturation.&nbsp;

In this publication, Winkler et al (2019) employed m6A -seq to demonstrate how m6A modifications on mRNA play a crucial role in controlling the innate immune response to infection, particularly by targeting type I interferons. This study underscores the significance of m6A-seq in elucidating the molecular mechanisms by which m6A modifications regulate the immune system, highlighting its importance in understanding and potentially manipulating immune responses to infections.

m6A-seq is pivotal in cancer research for identifying m6A modification patterns in mRNA that influence gene regulation and cancer progression. In their study, the researchers used m6A-seq to uncover differential m6A methylation in colorectal cancer, revealing potential targets for therapeutic intervention and understanding cancer mechanisms.&nbsp;

Since the first introduction of a m6A-seq protocol, a more recent and complex version was published, suggesting the m6A-SAC-seq approach (Ge et al, Nat Protoc 2023).&nbsp;This includes the chemical conversion of the methylated sites of interest by a dimethyl transferase to dimethyl adenosine (N6,6A), which then enables the use of the direct sequencing approach described before. Here, we will describe the steps of the classical m6A-seq protocol to keep things simple. These are the steps to successfully conduct a m6A-seq experiment (Dominissini et al, Nat Protoc 2013):&nbsp; 1. RNA isolation: For RNA isolation, any isolation method or kit can be used. It is important that the purified RNA is completely free of EDTA since it could interfere with the following fragmentation step.&nbsp;&nbsp;&nbsp;&nbsp; 2. RNA fragmentation: Metal-ion induced fragmentation is recommended for this step. The fragmentation should be calibrated beforehand to yield well reproducible results. Fragments of a size around 100 bp are recommended.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 3. Immunoprecipitation: This is the step that will pull down the RNAs containing a N6-methyladenosine site using a highly specific antibody bound to beads. Pulled RNAs will be bound to the beads in the end of this step. The use of RNase inhibitor is very important here.&nbsp; 4. Elution: For elution, two methods can be applied: Either competitive elution with free m6A or proteinase K digestion are possible ways to separate RNAs from the beads.&nbsp;&nbsp; 5. Amplification: The fused cDNA fragments are amplified.&nbsp; 6. Library preparation and massively parallel sequencing: Using a library preparation kit that matches the sequencing platform you are using; cDNA synthesis and adapter ligation yields a library ready to be sequenced.&nbsp; 7. Data Analysis: This crucial step is all about getting the most out of your experiment. Through processing your sequencing data, you can gain valuable biological insights.&nbsp; To get the best out of your m6A-seq data, we recommend outsourcing your data analysis to professional bioinformaticians. This way you can be sure the responsible person has the time and resources to always keep up to date with new developments in analysis methods while still understanding the biological background of the information you wish to obtain from your experiments.&nbsp;

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These are the steps our bioinformaticians will take to analyse your m6A-sequencing data:&nbsp; 1. Quality Control: Assessing the quality of raw sequencing reads to ensure high accuracy. This step is crucial for identifying and removing low-quality sequences that can affect downstream analysis.&nbsp; 2. Read Mapping: Alignment of raw sequencing reads to a reference genome or transcriptome. This step is essential for locating the reads within the genome and understanding their genomic context.&nbsp; 3. Peak Calling: Identifying regions with significant enrichment of m6A signals compared to a background model. This step will yield the positions of m6A methylation sites.&nbsp; 4. Filtering and Normalisation: This step is crucial and greatly influences the outcome of the data analysis. Data and experimental biases must be filtered as well as less abundant reads to gain valuable information from this big pack of data.&nbsp; 5. Differential Methylation Analysis: Comparison of methylation patterns across different conditions or samples to find changes. It is key to discovering how methylation varies in different biological states or diseases.&nbsp; 6. Functional Analysis: Investigate the biological significance of differentially methylated genes. It involves pathway and enrichment analyses to interpret the role of these modifications in cellular processes.&nbsp; 7. Visualisation: Plots typically used for this step are peak distribution histograms, enrichment plots and motif enrichment analysis plots.&nbsp;

What can be expected of next generation sequencing in the field of RNA modifications?

The development of novel RNA modification detection methods is crucial for advancing our understanding of their roles in biology and disease. However, challenges in mapping and quantifying these modifications hinder their full clinical application. Future research aims to improve the sensitivity and specificity of these methods, understand the regulatory networks of RNA modifications, and optimize RNA-based therapies and drugs targeting RNA-modifying proteins (Delaunay et al, Nat Rev Genet 2024).&nbsp;

For inquiries about integrating m6A-sequencing into your project or any related questions, we are here to assist. <a href="https://omiqa-backend.byintention.de/contact-us/">Contact us to explore how our expertise can complement your research needs.</a> Our team is excited to discuss potential collaborations and tailor our services to your specific requirements. Let’s connect and turn your scientific ideas into reality with cutting-edge RNA-seq applications!&nbsp;

Chen, J., Zhang, Y. C., Huang, C., Shen, H., Sun, B., Cheng, X., Zhang, Y. J., Yang, Y. G., Shu, Q., Yang, Y. &amp; Li, X. 2019. m(6)A Regulates Neurogenesis and Neuronal Development by Modulating Histone Methyltransferase Ezh2. Genomics Proteomics Bioinformatics, 17, 154-168.&nbsp; Delaunay, S., Helm, M. &amp; Frye, M. 2024. RNA modifications in physiology and disease: towards clinical applications. Nat Rev Genet, 25, 104-122.&nbsp; Dominissini, D., Moshitch-Moshkovitz, S., Salmon-Divon, M., Amariglio, N. &amp; Rechavi, G. 2013. Transcriptome-wide mapping of N(6)-methyladenosine by m(6)A-seq based on immunocapturing and massively parallel sequencing. Nat Protoc, 8, 176-89.&nbsp; Ge, R., Ye, C., Peng, Y., Dai, Q., Zhao, Y., Liu, S., Wang, P., Hu, L. &amp; He, C. 2023. m(6)A-SAC-seq for quantitative whole transcriptome m(6)A profiling. Nat Protoc, 18, 626-657.&nbsp; Li, S., Hu, W., Gong, S., Zhang, P., Cheng, J., Wang, S., Wang, Y., Shi, W., Li, Q., Wang, F. &amp; Yuan, Z. 2023. The Role of PRRC2B in Cerebral Vascular Remodeling Under Acute Hypoxia in Mice. Adv Sci (Weinh), 10, e2300892.&nbsp; Winkler, R., Gillis, E., Lasman, L., Safra, M., Geula, S., Soyris, C., Nachshon, A., Tai-Schmiedel, J., Friedman, N., Le-Trilling, V. T. K., Trilling, M., Mandelboim, M., Hanna, J. H., Schwartz, S. &amp; Stern-Ginossar, N. 2019. m(6)A modification controls the innate immune response to infection by targeting type I interferons. Nat Immunol, 20, 173-182.&nbsp;

Thursday’s technique: m6A-seq

Thursday’s technique: PRO-seq

Thursday’s technique: bulk RNA-seq

Thursday’s technique: ChIP-seq

Thursday’s technique: ATAC-seq

Published November 2023 in iScience, in collaboration with Medical University Innsbruck

Start with the basic building block of all narrative.

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Published November 2023 in iScience, in collaboration with Medical University Innsbruck

Non-coding RNAs (ncRNAs) are pivotal in gene regulation during development and disease. MicroRNAs have been extensively studied in neurogenesis. However, limited knowledge exists about the developmental signatures of other ncRNA species in sensory neuron differentiation, and human dorsal root ganglia (DRG) ncRNA expression remains undocumented. To address this gap, we generated a comprehensive atlas of small ncRNA species during iPSC-derived sensory neuron differentiation. Utilizing iPSC-derived sensory neurons and human DRG RNA sequencing, we unveiled signatures describing developmental processes. Our analysis identified ncRNAs associated with various sensory neuron stages. Striking similarities in ncRNA expression signatures between human DRG and iPSC-derived neurons support the latter as a model to bridge the translational gap between preclinical findings and human disorders. In summary, our research sheds light on the role of ncRNA species in human nociceptors, and NOCICEPTRA2.0 offers a comprehensive ncRNA database for sensory neurons that researchers can use to explore ncRNA regulators in nociceptors thoroughly.

Non-coding RNAs (ncRNAs) are pivotal in gene regulation during development and disease. MicroRNAs have been extensively studied in neurogenesis. However, limited knowledge exists about the developmental signatures of other ncRNA species in sensory neuron differentiation, and human dorsal root ganglia (DRG) ncRNA expression remains undocumented. To address this gap, we generated a comprehensive atlas of small ncRNA species during iPSC-derived sensory neuron differentiation. Utilizing iPSC-derived sensory neurons and human DRG RNA sequencing, we unveiled signatures describing developmental processes. Our analysis identified ncRNAs associated with various sensory neuron stages. Striking similarities in ncRNA expression signatures between human DRG and iPSC-derived neurons support the latter as a model to bridge the translational gap between preclinical findings and human disorders. In summary, our research sheds light on the role of ncRNA species in human nociceptors, and NOCICEPTRA2.0 offers a comprehensive ncRNA database for sensory neurons that researchers can use to explore ncRNA regulators in nociceptors thoroughly.

Check out the interactive application here: <a href="https://nociceptra.streamlit.app/" target="_blank" rel="noreferrer noopener">https://nociceptra.streamlit.app/</a> Read the full publication here: <a href="https://doi.org/10.1016/j.isci.2023.108525" target="_blank" rel="noreferrer noopener">https://doi.org/10.1016/j.isci.2023.108525</a>

Check out the interactive application here: <a href="https://nociceptra.streamlit.app/" target="_blank" rel="noreferrer noopener">https://nociceptra.streamlit.app/</a> Read the full publication here: <a href="https://doi.org/10.1016/j.isci.2023.108525" target="_blank" rel="noreferrer noopener">https://doi.org/10.1016/j.isci.2023.108525</a>

Omiqa author: Max Zeidler

Omiqa author: Max Zeidler

NOCICEPTRA2.0 – A comprehensive ncRNA atlas of human native and iPSC-derived sensory neurons

The emergence of definitive human haematopoietic stem cells (HSCs) during Carnegie Stages (CS) 14-17 in the aorta-gonad-mesonephros (AGM) region is a tightly regulated process. Previously, we conducted spatial transcriptomic analysis of the human AGM region at the end of this period (CS16/17) and identified secreted factors involved in HSC development. Here, we extend our analysis to investigate the progression of dorso-ventral polarized signalling around the dorsal aorta over the entire period of HSC emergence. Our results reveal a dramatic increase in ventral signalling complexity from the CS13 to CS14 transition, coinciding with the first appearance of definitive HSCs. We further observe stage-specific changes in signalling up to CS17 which may underpin the step-wise maturation of HSCs described in the mouse model. The data-rich resource is also presented in an online interface enabling in silico analysis of molecular interactions between spatially defined domains of the AGM region. This resource will be of particular interest for researchers studying mechanisms underlying human HSC development as well as those developing in vitro methods for the generation of clinically relevant HSCs from pluripotent stem cells.

The emergence of definitive human haematopoietic stem cells (HSCs) during Carnegie Stages (CS) 14-17 in the aorta-gonad-mesonephros (AGM) region is a tightly regulated process. Previously, we conducted spatial transcriptomic analysis of the human AGM region at the end of this period (CS16/17) and identified secreted factors involved in HSC development. Here, we extend our analysis to investigate the progression of dorso-ventral polarized signalling around the dorsal aorta over the entire period of HSC emergence. Our results reveal a dramatic increase in ventral signalling complexity from the CS13 to CS14 transition, coinciding with the first appearance of definitive HSCs. We further observe stage-specific changes in signalling up to CS17 which may underpin the step-wise maturation of HSCs described in the mouse model. The data-rich resource is also presented in an online interface enabling in silico analysis of molecular interactions between spatially defined domains of the AGM region. This resource will be of particular interest for researchers studying mechanisms underlying human HSC development as well as those developing in vitro methods for the generation of clinically relevant HSCs from pluripotent stem cells.

Check out the interactive application at <a href="https://medvinsky-lab.github.io/hsc-niche-explorer/" target="_blank" rel="noreferrer noopener">https://medvinsky-lab.github.io/hsc-niche-explorer/</a> Read the full publication here: <a href="https://doi.org/10.1242/dev.201972" target="_blank" rel="noreferrer noopener">https://doi.org/10.1242/dev.201972</a>

Check out the interactive application at <a href="https://medvinsky-lab.github.io/hsc-niche-explorer/" target="_blank" rel="noreferrer noopener">https://medvinsky-lab.github.io/hsc-niche-explorer/</a> Read the full publication here: <a href="https://doi.org/10.1242/dev.201972" target="_blank" rel="noreferrer noopener">https://doi.org/10.1242/dev.201972</a>

An interactive resource of molecular signalling in the developing human haematopoietic stem cell niche

Read the full publication here: <a href="https://doi.org/10.1016/j.gene.2023.147915" target="_blank" rel="noreferrer noopener">https://doi.org/10.1016/j.gene.2023.147915</a>

Read the full publication here: <a href="https://doi.org/10.1016/j.gene.2023.147915" target="_blank" rel="noreferrer noopener">https://doi.org/10.1016/j.gene.2023.147915</a>

S-adenosylmethionine treatment affects histone methylation in prostate cancer cells

Do you look for alternative splicing in your RNA-Sequencing data?

Published March 2023 in EMBO Molecular Medicine, in collaboration with Freie Universität Berlin and University of Cambridge.

Published March 2023 in EMBO Molecular Medicine, in collaboration with Freie Universität Berlin and University of Cambridge.

Neurodegenerative diseases are increasingly prevalent in the aging population, yet no disease-modifying treatments are currently available. Increasing the expression of the cold-shock protein RBM3 through therapeutic hypothermia is remarkably neuroprotective. However, systemic cooling poses a health risk, strongly limiting its clinical application. Selective upregulation of RBM3 at normothermia thus holds immense therapeutic potential. Here we identify a poison exon within the RBM3 gene that is solely responsible for its cold-induced expression. Genetic removal or antisense oligonucleotide (ASO)-mediated manipulation of this exon yields high RBM3 levels independent of cooling. Notably, a single administration of ASO to exclude the poison exon, using FDA-approved chemistry, results in long-lasting increased RBM3 expression in mouse brains. In prion-diseased mice, this treatment leads to remarkable neuroprotection, with prevention of neuronal loss and spongiosis despite high levels of disease-associated prion protein. Our promising results in mice support the possibility that RBM3-inducing ASOs might also deliver neuroprotection in humans in conditions ranging from acute brain injury to Alzheimer's disease.

Neurodegenerative diseases are increasingly prevalent in the aging population, yet no disease-modifying treatments are currently available. Increasing the expression of the cold-shock protein RBM3 through therapeutic hypothermia is remarkably neuroprotective. However, systemic cooling poses a health risk, strongly limiting its clinical application. Selective upregulation of RBM3 at normothermia thus holds immense therapeutic potential. Here we identify a poison exon within the RBM3 gene that is solely responsible for its cold-induced expression. Genetic removal or antisense oligonucleotide (ASO)-mediated manipulation of this exon yields high RBM3 levels independent of cooling. Notably, a single administration of ASO to exclude the poison exon, using FDA-approved chemistry, results in long-lasting increased RBM3 expression in mouse brains. In prion-diseased mice, this treatment leads to remarkable neuroprotection, with prevention of neuronal loss and spongiosis despite high levels of disease-associated prion protein. Our promising results in mice support the possibility that RBM3-inducing ASOs might also deliver neuroprotection in humans in conditions ranging from acute brain injury to Alzheimer's disease.

Read the full publication here: <a href="https://doi.org/10.15252/emmm.202217157" target="_blank" rel="noreferrer noopener">https://doi.org/10.15252/emmm.202217157</a>

Read the full publication here: <a href="https://doi.org/10.15252/emmm.202217157" target="_blank" rel="noreferrer noopener">https://doi.org/10.15252/emmm.202217157</a>

Omiqa authors: Tom Haltenhof &amp; Alexander Neumann

Omiqa authors: Tom Haltenhof &amp; Alexander Neumann

ASO targeting RBM3 temperature-controlled poison exon splicing prevents neurodegeneration in vivo

Published March 2023 in Science Advances, in collaboration with Freie Universität Berlin and the Lührmann group from MPI for Multidisciplinary Sciences Göttingen.

Published March 2023 in Science Advances, in collaboration with Freie Universität Berlin and the Lührmann group from MPI for Multidisciplinary Sciences Göttingen.

Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3′ splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA–mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spliceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3′ss. Cryo–electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage. They further elucidate the path of the 3′ region of the intron, allowing a structure-based model for how the C* spliceosome potentially scans for the proximal 3′ss. By combining biochemical and structural approaches with genome-wide functional analyses, our studies reveal widespread regulation of alternative 3′ss usage after step 1 of splicing and the likely mechanisms whereby C* proteins influence NAGNAG 3′ss choices.

Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3′ splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA–mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spliceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3′ss. Cryo–electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage. They further elucidate the path of the 3′ region of the intron, allowing a structure-based model for how the C* spliceosome potentially scans for the proximal 3′ss. By combining biochemical and structural approaches with genome-wide functional analyses, our studies reveal widespread regulation of alternative 3′ss usage after step 1 of splicing and the likely mechanisms whereby C* proteins influence NAGNAG 3′ss choices.

Read the full publication here: <a href="https://doi.org/10.1126/sciadv.adf1785" target="_blank" rel="noreferrer noopener">https://doi.org/10.1126/sciadv.adf1785</a>

Read the full publication here: <a href="https://doi.org/10.1126/sciadv.adf1785" target="_blank" rel="noreferrer noopener">https://doi.org/10.1126/sciadv.adf1785</a>

Omiqa contributer: Alexander Neumann

Omiqa contributer: Alexander Neumann

Regulation of 3′ splice site selection after step 1 of splicing by spliceosomal C* proteins

Published February 2023 in Biochemical and Biophysical Research Communications, in collaboration with Freie Universität Berlin.

Published February 2023 in Biochemical and Biophysical Research Communications, in collaboration with Freie Universität Berlin.

RNA-Seq has become the standard approach to quantify and compare gene expression and alternative splicing in different conditions. In many cases the limiting factor is not the sequencing itself but the bioinformatic analysis. A variety of software tools exist that predict alternative splicing patterns from RNA-Seq data, but surprisingly, a systematic comparison of the predictions obtained from different pipelines has not been performed. Here we compare results from frequently used bioinformatic tools using a high-quality RNA-Seq dataset. We show that there is little overlap in the splicing changes predicted by different tools and that GO-term analysis of the splicing changes predicted by the individual targets yields very different results. Validation of bioinformatic predictions by RT-PCR suggest a high number of false positives in the splicing changes predicated by each pipeline, which probably dominates GO-term analysis. The validation rate is strongly increased for targets predicted by several tools, offering a strategy to reduce false positives. Based on these results we offer some guidelines that may contribute to make alternative splicing predictions more reliable and may thus increase the impact of conclusions drawn from RNA-Seq studies. Furthermore, we created rmappet, a nextflow pipeline that performs alternative splicing analysis using rMATS and Whippet with subsequent overlapping of the results, enabling robust splicing analysis with only one command (<a href="https://github.com/didrikolofsson/rmappet/" target="_blank" rel="noreferrer noopener">https://github.com/didrikolofsson/rmappet/</a>).

RNA-Seq has become the standard approach to quantify and compare gene expression and alternative splicing in different conditions. In many cases the limiting factor is not the sequencing itself but the bioinformatic analysis. A variety of software tools exist that predict alternative splicing patterns from RNA-Seq data, but surprisingly, a systematic comparison of the predictions obtained from different pipelines has not been performed. Here we compare results from frequently used bioinformatic tools using a high-quality RNA-Seq dataset. We show that there is little overlap in the splicing changes predicted by different tools and that GO-term analysis of the splicing changes predicted by the individual targets yields very different results. Validation of bioinformatic predictions by RT-PCR suggest a high number of false positives in the splicing changes predicated by each pipeline, which probably dominates GO-term analysis. The validation rate is strongly increased for targets predicted by several tools, offering a strategy to reduce false positives. Based on these results we offer some guidelines that may contribute to make alternative splicing predictions more reliable and may thus increase the impact of conclusions drawn from RNA-Seq studies. Furthermore, we created rmappet, a nextflow pipeline that performs alternative splicing analysis using rMATS and Whippet with subsequent overlapping of the results, enabling robust splicing analysis with only one command (<a href="https://github.com/didrikolofsson/rmappet/" target="_blank" rel="noreferrer noopener">https://github.com/didrikolofsson/rmappet/</a>).

Read the full publication here: <a href="https://doi.org/10.1016/j.bbrc.2023.02.053" target="_blank" rel="noreferrer noopener">https://doi.org/10.1016/j.bbrc.2023.02.053</a>

Read the full publication here: <a href="https://doi.org/10.1016/j.bbrc.2023.02.053" target="_blank" rel="noreferrer noopener">https://doi.org/10.1016/j.bbrc.2023.02.053</a>

Omiqa authors: Alexander Neumann &amp; Didrik Olofsson

Omiqa authors: Alexander Neumann &amp; Didrik Olofsson

One pipeline to predict them all? On the prediction of alternative splicing from RNA-Seq data

Published December 2022 in Life Science Alliance, in collaboration with Freie Universität Berlin.

Published December 2022 in Life Science Alliance, in collaboration with Freie Universität Berlin.

Species-specific regulation and functionality of alternative splicing remained unknown to date. Calcium/calmodulin-dependent protein kinase IIβ (CaMKIIβ) is expressed in several splice variants and plays a key role in learning and memory. In the present study, we investigated and characterised several primate-specific CAMK2B splice isoforms. These isoforms show altered kinetic properties and changes in substrate specificity. Moreover we demonstrate branch point weakening as a cause of altered splicing during evolution. Reducing branch point and splice site strengths globally renders constitutive exons alternative, thus providing novel mechanistic insight into cis-directed species-specific alternative splicing regulation. Using CRISPR/Cas9, we inserted a weaker, human branch point sequence into the mouse genome and observed strongly altered Camk2b splicing events within the brains of mutant mice. We observe a strong impairment of long-term potentiation in CA3-CA1 synapses of mutant mice. In summary we connect branch point–controlled CAMK2B alternative splicing with a fundamental function in learning and memory.

Species-specific regulation and functionality of alternative splicing remained unknown to date. Calcium/calmodulin-dependent protein kinase IIβ (CaMKIIβ) is expressed in several splice variants and plays a key role in learning and memory. In the present study, we investigated and characterised several primate-specific CAMK2B splice isoforms. These isoforms show altered kinetic properties and changes in substrate specificity. Moreover we demonstrate branch point weakening as a cause of altered splicing during evolution. Reducing branch point and splice site strengths globally renders constitutive exons alternative, thus providing novel mechanistic insight into cis-directed species-specific alternative splicing regulation. Using CRISPR/Cas9, we inserted a weaker, human branch point sequence into the mouse genome and observed strongly altered Camk2b splicing events within the brains of mutant mice. We observe a strong impairment of long-term potentiation in CA3-CA1 synapses of mutant mice. In summary we connect branch point–controlled CAMK2B alternative splicing with a fundamental function in learning and memory.

Read the full publication here: <a href="https://doi.org/10.26508/lsa.202201826" target="_blank" rel="noreferrer noopener">https://doi.org/10.26508/lsa.202201826</a>

Read the full publication here: <a href="https://doi.org/10.26508/lsa.202201826" target="_blank" rel="noreferrer noopener">https://doi.org/10.26508/lsa.202201826</a>

Omiqa author: Alexander Neumann

Omiqa author: Alexander Neumann

Branch point strength controls species-specific CAMK2B alternative splicing and regulates LTP

Published July 2022 in Nucleic Acids Research, in collaboration with Freie Universität Berlin.

Published July 2022 in Nucleic Acids Research, in collaboration with Freie Universität Berlin.

Antiviral innate immunity represents the first defense against invading viruses and is key to control viral infections, including SARS-CoV-2. Body temperature is an omnipresent variable but was neglected when addressing host defense mechanisms and susceptibility to SARS-CoV-2 infection. Here, we show that increasing temperature in a 1.5°C window, between 36.5 and 38°C, strongly increases the expression of genes in two branches of antiviral immunity, nitric oxide production and type I interferon response. We show that alternative splicing coupled to nonsense-mediated decay decreases STAT2 expression in colder conditions and suggest that increased STAT2 expression at elevated temperature induces the expression of diverse antiviral genes and SARS-CoV-2 restriction factors. This cascade is activated in a remarkably narrow temperature range below febrile temperature, which reflects individual, circadian and age-dependent variation. We suggest that decreased body temperature with aging contributes to reduced expression of antiviral genes in older individuals. Using cell culture and in vivo models, we show that higher body temperature correlates with reduced SARS-CoV-2 replication, which may affect the different vulnerability of children versus seniors toward severe SARS-CoV-2 infection. Altogether, our data connect body temperature and pre-mRNA processing to provide new mechanistic insight into the regulation of antiviral innate immunity.

Antiviral innate immunity represents the first defense against invading viruses and is key to control viral infections, including SARS-CoV-2. Body temperature is an omnipresent variable but was neglected when addressing host defense mechanisms and susceptibility to SARS-CoV-2 infection. Here, we show that increasing temperature in a 1.5°C window, between 36.5 and 38°C, strongly increases the expression of genes in two branches of antiviral immunity, nitric oxide production and type I interferon response. We show that alternative splicing coupled to nonsense-mediated decay decreases STAT2 expression in colder conditions and suggest that increased STAT2 expression at elevated temperature induces the expression of diverse antiviral genes and SARS-CoV-2 restriction factors. This cascade is activated in a remarkably narrow temperature range below febrile temperature, which reflects individual, circadian and age-dependent variation. We suggest that decreased body temperature with aging contributes to reduced expression of antiviral genes in older individuals. Using cell culture and in vivo models, we show that higher body temperature correlates with reduced SARS-CoV-2 replication, which may affect the different vulnerability of children versus seniors toward severe SARS-CoV-2 infection. Altogether, our data connect body temperature and pre-mRNA processing to provide new mechanistic insight into the regulation of antiviral innate immunity.

Read the full publication here: <a href="https://doi.org/10.1093/nar/gkac513" target="_blank" rel="noreferrer noopener">https://doi.org/10.1093/nar/gkac513</a>

Read the full publication here: <a href="https://doi.org/10.1093/nar/gkac513" target="_blank" rel="noreferrer noopener">https://doi.org/10.1093/nar/gkac513</a>

Omiqa author: Didrik Olofsson

Omiqa author: Didrik Olofsson

Body temperature variation controls pre-mRNA processing and transcription of antiviral genes and SARS-CoV-2 replication

Published June 2022 in Genetics in Medicine, in collaboration with Charité Universitätsmedizin Berlin.

Published June 2022 in Genetics in Medicine, in collaboration with Charité Universitätsmedizin Berlin.

We identified a lipodystrophy phenotype with a typical facial appearance, corneal clouding, achalasia, progressive hearing loss, and variable severity. Although 3 individuals showed stunted growth, intellectual disability, and died within the first decade of life (A1, A2, and A3), 2 are adults with normal intellectual development (A4 and A5). All individuals harbored an identical homozygous nonsense variant affecting the retention and splicing complex component BUD13. The nucleotide substitution caused alternative splicing of BUD13 leading to a stable truncated protein whose expression positively correlated with disease expression and life expectancy. In dermal fibroblasts, we found elevated intron retention, a global reduction of spliceosomal proteins, and nuclei with multiple invaginations, which were more pronounced in A1, A2, and A3. Overexpression of both BUD13 isoforms normalized the nuclear morphology.

We identified a lipodystrophy phenotype with a typical facial appearance, corneal clouding, achalasia, progressive hearing loss, and variable severity. Although 3 individuals showed stunted growth, intellectual disability, and died within the first decade of life (A1, A2, and A3), 2 are adults with normal intellectual development (A4 and A5). All individuals harbored an identical homozygous nonsense variant affecting the retention and splicing complex component BUD13. The nucleotide substitution caused alternative splicing of BUD13 leading to a stable truncated protein whose expression positively correlated with disease expression and life expectancy. In dermal fibroblasts, we found elevated intron retention, a global reduction of spliceosomal proteins, and nuclei with multiple invaginations, which were more pronounced in A1, A2, and A3. Overexpression of both BUD13 isoforms normalized the nuclear morphology.

Read the full publication here: <a href="https://doi.org/10.1016/j.gim.2022.05.004" target="_blank" rel="noreferrer noopener">https://doi.org/10.1016/j.gim.2022.05.004</a>

Read the full publication here: <a href="https://doi.org/10.1016/j.gim.2022.05.004" target="_blank" rel="noreferrer noopener">https://doi.org/10.1016/j.gim.2022.05.004</a>

Omiqa author: Alexander Neumann

Alternative splicing of BUD13 determines the severity of a developmental disorder with lipodystrophy and progeroid features

Published June 2022 in Science of the Total Environment, in collaboration with Duke University.

Published June 2022 in Science of the Total Environment, in collaboration with Duke University.

Mixtures of per- and polyfluoroalkyl substances (PFAS) are often found in drinking water, and serum PFAS are detected in up to 99% of the population. However, very little is known about how exposure to mixtures of PFAS affects maternal and fetal health. The aim of this study was to investigate maternal, fetal, and placental outcomes after preconceptional and gestational exposure to an environmentally relevant PFAS mixture in a New Zealand White (NZW) rabbit model. Dams were exposed via drinking water to control (no detectable PFAS) or a PFAS mixture for 32 days. This mixture was formulated with PFAS to resemble levels measured in tap water from Pittsboro, NC (10 PFAS compounds; total PFAS load = 758.6 ng/L). Maternal, fetal, and placental outcomes were evaluated at necropsy. Thyroid hormones were measured in maternal serum and kit blood. Placental gene expression was evaluated by RNAseq and qPCR. PFAS exposure resulted in higher body weight (p = 0.01), liver (p = 0.01) and kidney (p = 0.01) weights, blood pressure (p = 0.05), and BUN:CRE ratio (p = 0.04) in dams, along with microscopic changes in renal cortices. Fetal weight, measures, and histopathology were unchanged, but a significant interaction between dose and sex was detected in the fetal: placental weight ratio (p = 0.036). Placental macroscopic changes were present in PFAS-exposed dams. Dam serum showed lower T4 and a higher T3:T4 ratio, although not statistically significant. RNAseq revealed that 11 of the 14 differentially expressed genes (adj. p &lt; 0.1) are involved in placentation or pregnancy complications. In summary, exposure elicited maternal weight gain and signs of hypertension, renal injury, sex-specific changes in placental response, and differential expression of genes involved in placentation and preeclampsia. Importantly, these are the first results to show adverse maternal and placental effects of an environmentally-relevant PFAS mixture in vivo.

Mixtures of per- and polyfluoroalkyl substances (PFAS) are often found in drinking water, and serum PFAS are detected in up to 99% of the population. However, very little is known about how exposure to mixtures of PFAS affects maternal and fetal health. The aim of this study was to investigate maternal, fetal, and placental outcomes after preconceptional and gestational exposure to an environmentally relevant PFAS mixture in a New Zealand White (NZW) rabbit model. Dams were exposed via drinking water to control (no detectable PFAS) or a PFAS mixture for 32 days. This mixture was formulated with PFAS to resemble levels measured in tap water from Pittsboro, NC (10 PFAS compounds; total PFAS load = 758.6 ng/L). Maternal, fetal, and placental outcomes were evaluated at necropsy. Thyroid hormones were measured in maternal serum and kit blood. Placental gene expression was evaluated by RNAseq and qPCR. PFAS exposure resulted in higher body weight (p = 0.01), liver (p = 0.01) and kidney (p = 0.01) weights, blood pressure (p = 0.05), and BUN:CRE ratio (p = 0.04) in dams, along with microscopic changes in renal cortices. Fetal weight, measures, and histopathology were unchanged, but a significant interaction between dose and sex was detected in the fetal: placental weight ratio (p = 0.036). Placental macroscopic changes were present in PFAS-exposed dams. Dam serum showed lower T4 and a higher T3:T4 ratio, although not statistically significant. RNAseq revealed that 11 of the 14 differentially expressed genes (adj. p &lt; 0.1) are involved in placentation or pregnancy complications. In summary, exposure elicited maternal weight gain and signs of hypertension, renal injury, sex-specific changes in placental response, and differential expression of genes involved in placentation and preeclampsia. Importantly, these are the first results to show adverse maternal and placental effects of an environmentally-relevant PFAS mixture in vivo.

Read the full publication here: <a href="https://doi.org/10.1016/j.scitotenv.2022.156499" target="_blank" rel="noreferrer noopener">https://doi.org/10.1016/j.scitotenv.2022.156499</a>

Read the full publication here: <a href="https://doi.org/10.1016/j.scitotenv.2022.156499" target="_blank" rel="noreferrer noopener">https://doi.org/10.1016/j.scitotenv.2022.156499</a>

Evaluating maternal exposure to an environmental per and polyfluoroalkyl substances (PFAS) mixture during pregnancy: Adverse maternal and fetoplacental effects in a New Zealand White (NZW) rabbit model

Published May 2022 in Gene Therapy, in collaboration with Touchlight Genetics.

Published May 2022 in Gene Therapy, in collaboration with Touchlight Genetics.

Traditional bacterial fermentation techniques used to manufacture plasmid are time-consuming, expensive, and inherently unstable. The production of sufficient GMP grade material thus imposes a major bottleneck on industrial-scale manufacturing of lentiviral vectors (LVV). Touchlight's linear doggybone DNA (dbDNATM) is an enzymatically amplified DNA vector produced with exceptional speed through an in vitro dual enzyme process, enabling industrial-scale manufacturing of GMP material in a fraction of the time required for plasmid. We have previously shown that dbDNATM can be used to produce functional LVV; however, obtaining high LVV titres remained a challenge. Here, we aimed to demonstrate that dbDNATM could be optimised for the manufacture of high titre LVV. We found that dbDNATM displayed a unique transfection and expression profile in the context of LVV production, which necessitated the optimisation of DNA input and construct ratios. Furthermore, we demonstrate that efficient 3' end processing of viral genomic RNA (vgRNA) derived from linear dbDNATM transfer vectors required the addition of a strong 3' termination signal and downstream spacer sequence to enable efficient vgRNA packaging. Using these improved vector architectures along with optimised transfection conditions, we were able to produce a CAR19h28z LVV with equivalent infectious titres as achieved using plasmid, demonstrating that dbDNATM technology can provide a highly effective solution to the plasmid bottleneck.

Traditional bacterial fermentation techniques used to manufacture plasmid are time-consuming, expensive, and inherently unstable. The production of sufficient GMP grade material thus imposes a major bottleneck on industrial-scale manufacturing of lentiviral vectors (LVV). Touchlight's linear doggybone DNA (dbDNATM) is an enzymatically amplified DNA vector produced with exceptional speed through an in vitro dual enzyme process, enabling industrial-scale manufacturing of GMP material in a fraction of the time required for plasmid. We have previously shown that dbDNATM can be used to produce functional LVV; however, obtaining high LVV titres remained a challenge. Here, we aimed to demonstrate that dbDNATM could be optimised for the manufacture of high titre LVV. We found that dbDNATM displayed a unique transfection and expression profile in the context of LVV production, which necessitated the optimisation of DNA input and construct ratios. Furthermore, we demonstrate that efficient 3' end processing of viral genomic RNA (vgRNA) derived from linear dbDNATM transfer vectors required the addition of a strong 3' termination signal and downstream spacer sequence to enable efficient vgRNA packaging. Using these improved vector architectures along with optimised transfection conditions, we were able to produce a CAR19h28z LVV with equivalent infectious titres as achieved using plasmid, demonstrating that dbDNATM technology can provide a highly effective solution to the plasmid bottleneck.

Read the full publication here: <a href="https://doi.org/10.1038/s41434-022-00343-4" target="_blank" rel="noreferrer noopener">https://doi.org/10.1038/s41434-022-00343-4</a>

Read the full publication here: <a href="https://doi.org/10.1038/s41434-022-00343-4" target="_blank" rel="noreferrer noopener">https://doi.org/10.1038/s41434-022-00343-4</a>

Omiqa authors: Didrik Olofsson and Alexander Neumann

Omiqa authors: Didrik Olofsson and Alexander Neumann

Enzymatically amplified linear dbDNA as a rapid and scalable solution to industrial lentiviral vector manufacturing

Published August 2021 in the International Journal of Molecular Sciences, in collaboration with the German Institute of Human Nutrition.

Published August 2021 in the International Journal of Molecular Sciences, in collaboration with the German Institute of Human Nutrition.

Dysfunctional islets of Langerhans are a hallmark of type 2 diabetes (T2D). We hypothesize that differences in islet gene expression alternative splicing which can contribute to altered protein function also participate in islet dysfunction. RNA sequencing (RNAseq) data from islets of obese diabetes-resistant and diabetes-susceptible mice were analyzed for alternative splicing and its putative genetic and epigenetic modulators. We focused on the expression levels of chromatin modifiers and SNPs in regulatory sequences. We identified alternative splicing events in islets of diabetes-susceptible mice amongst others in genes linked to insulin secretion, endocytosis or ubiquitin-mediated proteolysis pathways. The expression pattern of 54 histones and chromatin modifiers, which may modulate splicing, were markedly downregulated in islets of diabetic animals. Furthermore, diabetes-susceptible mice carry SNPs in RNA-binding protein motifs and in splice sites potentially responsible for alternative splicing events. They also exhibit a larger exon skipping rate, e.g., in the diabetes gene Abcc8, which might affect protein function. Expression of the neuronal splicing factor Srrm4 which mediates inclusion of microexons in mRNA transcripts was markedly lower in islets of diabetes-prone compared to diabetes-resistant mice, correlating with a preferential skipping of SRRM4 target exons. The repression of Srrm4 expression is presumably mediated via a higher expression of miR-326-3p and miR-3547-3p in islets of diabetic mice. Thus, our study suggests that an altered splicing pattern in islets of diabetes-susceptible mice may contribute to an elevated T2D risk.

Dysfunctional islets of Langerhans are a hallmark of type 2 diabetes (T2D). We hypothesize that differences in islet gene expression alternative splicing which can contribute to altered protein function also participate in islet dysfunction. RNA sequencing (RNAseq) data from islets of obese diabetes-resistant and diabetes-susceptible mice were analyzed for alternative splicing and its putative genetic and epigenetic modulators. We focused on the expression levels of chromatin modifiers and SNPs in regulatory sequences. We identified alternative splicing events in islets of diabetes-susceptible mice amongst others in genes linked to insulin secretion, endocytosis or ubiquitin-mediated proteolysis pathways. The expression pattern of 54 histones and chromatin modifiers, which may modulate splicing, were markedly downregulated in islets of diabetic animals. Furthermore, diabetes-susceptible mice carry SNPs in RNA-binding protein motifs and in splice sites potentially responsible for alternative splicing events. They also exhibit a larger exon skipping rate, e.g., in the diabetes gene Abcc8, which might affect protein function. Expression of the neuronal splicing factor Srrm4 which mediates inclusion of microexons in mRNA transcripts was markedly lower in islets of diabetes-prone compared to diabetes-resistant mice, correlating with a preferential skipping of SRRM4 target exons. The repression of Srrm4 expression is presumably mediated via a higher expression of miR-326-3p and miR-3547-3p in islets of diabetic mice. Thus, our study suggests that an altered splicing pattern in islets of diabetes-susceptible mice may contribute to an elevated T2D risk.

Read the full publication here: <a href="https://doi.org/10.3390/ijms22168597" target="_blank" rel="noreferrer noopener">https://doi.org/10.3390/ijms22168597</a>

Read the full publication here: <a href="https://doi.org/10.3390/ijms22168597" target="_blank" rel="noreferrer noopener">https://doi.org/10.3390/ijms22168597</a>

Enriched Alternative Splicing in Islets of Diabetes-Susceptible Mice

Published January 2021 in Neuron, in collaboration with Rady Children's Institute for Genomic Medicine (San Diego).

Published January 2021 in Neuron, in collaboration with Rady Children's Institute for Genomic Medicine (San Diego).

Autosomal-recessive cerebellar hypoplasia and ataxia constitute a group of heterogeneous brain disorders caused by disruption of several fundamental cellular processes. Here, we identified 10 families showing a neurodegenerative condition involving pontocerebellar hypoplasia with microcephaly (PCHM). Patients harbored biallelic mutations in genes encoding the spliceosome components Peptidyl-Prolyl Isomerase Like-1 (PPIL1) or Pre-RNA Processing-17 (PRP17). Mouse knockouts of either gene were lethal in early embryogenesis, whereas PPIL1 patient mutation knockin mice showed neuron-specific apoptosis. Loss of either protein affected splicing integrity, predominantly affecting short and high GC-content introns and genes involved in brain disorders. PPIL1 and PRP17 form an active isomerase-substrate interaction, but we found that isomerase activity is not critical for function. Thus, we establish disrupted splicing integrity and "major spliceosome-opathies" as a new mechanism underlying PCHM and neurodegeneration and uncover a non-enzymatic function of a spliceosomal proline isomerase.

Autosomal-recessive cerebellar hypoplasia and ataxia constitute a group of heterogeneous brain disorders caused by disruption of several fundamental cellular processes. Here, we identified 10 families showing a neurodegenerative condition involving pontocerebellar hypoplasia with microcephaly (PCHM). Patients harbored biallelic mutations in genes encoding the spliceosome components Peptidyl-Prolyl Isomerase Like-1 (PPIL1) or Pre-RNA Processing-17 (PRP17). Mouse knockouts of either gene were lethal in early embryogenesis, whereas PPIL1 patient mutation knockin mice showed neuron-specific apoptosis. Loss of either protein affected splicing integrity, predominantly affecting short and high GC-content introns and genes involved in brain disorders. PPIL1 and PRP17 form an active isomerase-substrate interaction, but we found that isomerase activity is not critical for function. Thus, we establish disrupted splicing integrity and "major spliceosome-opathies" as a new mechanism underlying PCHM and neurodegeneration and uncover a non-enzymatic function of a spliceosomal proline isomerase.

Read the full publication here: <a href="https://doi.org/10.1016/j.neuron.2020.10.035" target="_blank" rel="noreferrer noopener">https://doi.org/10.1016/j.neuron.2020.10.035</a>

Read the full publication here: <a href="https://doi.org/10.1016/j.neuron.2020.10.035" target="_blank" rel="noreferrer noopener">https://doi.org/10.1016/j.neuron.2020.10.035</a>

Mutations in Spliceosomal Genes PPIL1 and PRP17 Cause Neurodegenerative Pontocerebellar Hypoplasia with Microcephaly

Published December 2020 in EMBO Reports, in collaboration with Freie Universität Berlin.

Published December 2020 in EMBO Reports, in collaboration with Freie Universität Berlin.

Mammalian body temperature oscillates with the time of the day and is altered in diverse pathological conditions. We recently identified a body temperature-sensitive thermometer-like kinase, which alters SR protein phosphorylation and thereby globally controls alternative splicing (AS). AS can generate unproductive variants which are recognized and degraded by diverse mRNA decay pathways—including nonsense-mediated decay (NMD). Here we show extensive coupling of body temperature-controlled AS to mRNA decay, leading to global control of temperature-dependent gene expression (GE). Temperature-controlled, decay-inducing splicing events are evolutionarily conserved and pervasively found within RNA-binding proteins, including most SR proteins. AS-coupled poison exon inclusion is essential for rhythmic GE of SR proteins and has a global role in establishing temperature-dependent rhythmic GE profiles, both in mammals under circadian body temperature cycles and in plants in response to ambient temperature changes. Together, these data identify body temperature-driven AS-coupled mRNA decay as an evolutionary ancient, core clock-independent mechanism to generate rhythmic GE.

Mammalian body temperature oscillates with the time of the day and is altered in diverse pathological conditions. We recently identified a body temperature-sensitive thermometer-like kinase, which alters SR protein phosphorylation and thereby globally controls alternative splicing (AS). AS can generate unproductive variants which are recognized and degraded by diverse mRNA decay pathways—including nonsense-mediated decay (NMD). Here we show extensive coupling of body temperature-controlled AS to mRNA decay, leading to global control of temperature-dependent gene expression (GE). Temperature-controlled, decay-inducing splicing events are evolutionarily conserved and pervasively found within RNA-binding proteins, including most SR proteins. AS-coupled poison exon inclusion is essential for rhythmic GE of SR proteins and has a global role in establishing temperature-dependent rhythmic GE profiles, both in mammals under circadian body temperature cycles and in plants in response to ambient temperature changes. Together, these data identify body temperature-driven AS-coupled mRNA decay as an evolutionary ancient, core clock-independent mechanism to generate rhythmic GE.

Read the full publication here: <a href="https://doi.org/10.15252/embr.202051369" target="_blank" rel="noreferrer noopener">https://doi.org/10.15252/embr.202051369</a>

Read the full publication here: <a href="https://doi.org/10.15252/embr.202051369" target="_blank" rel="noreferrer noopener">https://doi.org/10.15252/embr.202051369</a>

Alternative splicing coupled mRNA decay shapes the temperature-dependent transcriptome

Published October 2020 in Journal of Hepatology, in collaboration with the German Institute of Human Nutrition.

Published October 2020 in Journal of Hepatology, in collaboration with the German Institute of Human Nutrition.

The genetic basis of non-alcoholic fatty liver disease remains incompletely defined. Herein, we identified members of the immunity-related GTPase family in mice and humans that act as regulators of hepatic fat accumulation, with links to autophagy. Overexpression of the gene Ifgga2 was shown to reduce hepatic lipid storage and could be a therapeutic target for the treatment of fatty liver disease.

The genetic basis of non-alcoholic fatty liver disease remains incompletely defined. Herein, we identified members of the immunity-related GTPase family in mice and humans that act as regulators of hepatic fat accumulation, with links to autophagy. Overexpression of the gene Ifgga2 was shown to reduce hepatic lipid storage and could be a therapeutic target for the treatment of fatty liver disease.

Read the full publication here: <a href="https://doi.org/10.1016/j.jhep.2020.04.031" target="_blank" rel="noreferrer noopener">https://doi.org/10.1016/j.jhep.2020.04.031</a>

Read the full publication here: <a href="https://doi.org/10.1016/j.jhep.2020.04.031" target="_blank" rel="noreferrer noopener">https://doi.org/10.1016/j.jhep.2020.04.031</a>

Omiqa author: Tom Haltenhof

Omiqa author: Tom Haltenhof

Immunity-related GTPase induces lipophagy to prevent excess hepatic lipid accumulation

Published June 2019 in RNA Biology, in collaboration with Freie Universität Berlin.

Published June 2019 in RNA Biology, in collaboration with Freie Universität Berlin.

Recent work has associated point mutations in both zinc fingers (ZnF) of the spliceosome component U2AF35 with malignant transformation. However, surprisingly little is known about the functionality of the U2AF35 ZnF domains in general. Here we have analysed key functionalities of the ZnF domains of mammalian U2AF35 and its paralog U2AF26. Both ZnFs are required for splicing regulation, whereas only ZnF2 controls protein stability and contributes to the interaction with U2AF65. These features are confirmed in a naturally occurring splice variant of U2AF26 lacking ZnF2, that is strongly induced upon activation of primary mouse T cells and localized in the cytoplasm. Using Ribo-Seq in a model T cell line we provide evidence for a role of U2AF26 in activating cytoplasmic steps in gene expression, notably translation. Consistently, an MS2 tethering assay shows that cytoplasmic U2AF26/35 increase translation when localized to the 5'UTR of a model mRNA. This regulation is partially dependent on ZnF1 thus providing a connection between a core splicing factor, the ZnF domains and the regulation of translation. Altogether, our work reveals unexpected functions of U2AF26/35 and their ZnF domains, thereby contributing to a better understanding of their role and regulation in mammalian cells.

Recent work has associated point mutations in both zinc fingers (ZnF) of the spliceosome component U2AF35 with malignant transformation. However, surprisingly little is known about the functionality of the U2AF35 ZnF domains in general. Here we have analysed key functionalities of the ZnF domains of mammalian U2AF35 and its paralog U2AF26. Both ZnFs are required for splicing regulation, whereas only ZnF2 controls protein stability and contributes to the interaction with U2AF65. These features are confirmed in a naturally occurring splice variant of U2AF26 lacking ZnF2, that is strongly induced upon activation of primary mouse T cells and localized in the cytoplasm. Using Ribo-Seq in a model T cell line we provide evidence for a role of U2AF26 in activating cytoplasmic steps in gene expression, notably translation. Consistently, an MS2 tethering assay shows that cytoplasmic U2AF26/35 increase translation when localized to the 5'UTR of a model mRNA. This regulation is partially dependent on ZnF1 thus providing a connection between a core splicing factor, the ZnF domains and the regulation of translation. Altogether, our work reveals unexpected functions of U2AF26/35 and their ZnF domains, thereby contributing to a better understanding of their role and regulation in mammalian cells.

Read the full publication here: <a href="https://doi.org/10.1080/15476286.2020.1732701" target="_blank" rel="noreferrer noopener">https://doi.org/10.1080/15476286.2020.1732701</a>

Read the full publication here: <a href="https://doi.org/10.1080/15476286.2020.1732701" target="_blank" rel="noreferrer noopener">https://doi.org/10.1080/15476286.2020.1732701</a>

The zinc finger domains in U2AF26 and U2AF35 have diverse functionalities including a role in controlling translation

Published April 2020 in Elife, in collaboration with Freie Universität Berlin.

Published April 2020 in Elife, in collaboration with Freie Universität Berlin.

Minor and major spliceosomes control splicing of distinct intron types and are thought to act largely independent of one another. SR proteins are essential splicing regulators mostly connected to the major spliceosome. Here, we show that Srsf10 expression is controlled through an autoregulated minor intron, tightly correlating Srsf10 with minor spliceosome abundance across different tissues and differentiation stages in mammals. Surprisingly, all other SR proteins also correlate with the minor spliceosome and Srsf10, and abolishing Srsf10 autoregulation by Crispr/Cas9-mediated deletion of the autoregulatory exon induces expression of all SR proteins in a human cell line. Our data thus reveal extensive crosstalk and a global impact of the minor spliceosome on major intron splicing.

Minor and major spliceosomes control splicing of distinct intron types and are thought to act largely independent of one another. SR proteins are essential splicing regulators mostly connected to the major spliceosome. Here, we show that Srsf10 expression is controlled through an autoregulated minor intron, tightly correlating Srsf10 with minor spliceosome abundance across different tissues and differentiation stages in mammals. Surprisingly, all other SR proteins also correlate with the minor spliceosome and Srsf10, and abolishing Srsf10 autoregulation by Crispr/Cas9-mediated deletion of the autoregulatory exon induces expression of all SR proteins in a human cell line. Our data thus reveal extensive crosstalk and a global impact of the minor spliceosome on major intron splicing.

Read the full publication here: <a href="https://doi.org/10.7554/eLife.56075" target="_blank" rel="noreferrer noopener">https://doi.org/10.7554/eLife.56075</a>

Read the full publication here: <a href="https://doi.org/10.7554/eLife.56075" target="_blank" rel="noreferrer noopener">https://doi.org/10.7554/eLife.56075</a>

Srsf10 and the minor spliceosome control tissue-specific and dynamic SR protein expression

Published April 2020 in Molecular Cell, in collaboration with Freie Universität Berlin.

Published April 2020 in Molecular Cell, in collaboration with Freie Universität Berlin.

Homeothermic organisms maintain their core body temperature in a narrow, tightly controlled range. Whether and how subtle circadian oscillations or disease-associated changes in core body temperature are sensed and integrated in gene expression programs remain elusive. Furthermore, a thermo-sensor capable of sensing the small temperature differentials leading to temperature-dependent sex determination (TSD) in poikilothermic reptiles has not been identified. Here, we show that the activity of CDC-like kinases (CLKs) is highly responsive to physiological temperature changes, which is conferred by structural rearrangements within the kinase activation segment. Lower body temperature activates CLKs resulting in strongly increased phosphorylation of SR proteins&nbsp;in&nbsp;vitro&nbsp;and&nbsp;in&nbsp;vivo. This globally controls temperature-dependent alternative splicing and gene expression, with wide implications in circadian, tissue-specific, and disease-associated settings. This temperature sensor is conserved across evolution and adapted to growth temperatures of diverse poikilotherms. The dynamic temperature range of reptilian CLK homologs suggests a role in TSD.

Homeothermic organisms maintain their core body temperature in a narrow, tightly controlled range. Whether and how subtle circadian oscillations or disease-associated changes in core body temperature are sensed and integrated in gene expression programs remain elusive. Furthermore, a thermo-sensor capable of sensing the small temperature differentials leading to temperature-dependent sex determination (TSD) in poikilothermic reptiles has not been identified. Here, we show that the activity of CDC-like kinases (CLKs) is highly responsive to physiological temperature changes, which is conferred by structural rearrangements within the kinase activation segment. Lower body temperature activates CLKs resulting in strongly increased phosphorylation of SR proteins&nbsp;in&nbsp;vitro&nbsp;and&nbsp;in&nbsp;vivo. This globally controls temperature-dependent alternative splicing and gene expression, with wide implications in circadian, tissue-specific, and disease-associated settings. This temperature sensor is conserved across evolution and adapted to growth temperatures of diverse poikilotherms. The dynamic temperature range of reptilian CLK homologs suggests a role in TSD.

Read the full publication here: <a href="https://doi.org/10.1016/j.molcel.2020.01.028" target="_blank" rel="noreferrer noopener">https://doi.org/10.1016/j.molcel.2020.01.028</a>

Read the full publication here: <a href="https://doi.org/10.1016/j.molcel.2020.01.028" target="_blank" rel="noreferrer noopener">https://doi.org/10.1016/j.molcel.2020.01.028</a>

A Conserved Kinase-Based Body-Temperature Sensor Globally Controls Alternative Splicing and Gene Expression

Published July 2019 in Molecular and Cellular Biology, in collaboration with Freie Universität Berlin.

Published July 2019 in Molecular and Cellular Biology, in collaboration with Freie Universität Berlin.

T cells are nodal players in the adaptive immune response against pathogens and malignant cells. Alternative splicing plays a crucial role in T cell activation, which is analyzed mainly at later time points upon stimulation. Here we have discovered a 2-h time window early after stimulation where optimal splicing efficiency or, more generally, gene expression efficiency is crucial for successful T cell activation. Reducing the splicing efficiency at 4 to 6 h poststimulation significantly impaired murine T cell activation, which was dependent on the expression dynamics of the Egr1-Nab2-interleukin-2 (IL-2) pathway. This time window overlaps the time of peak IL-2&nbsp;de novo&nbsp;transcription, which, we suggest, represents a permissive time window in which decreased splicing (or transcription) efficiency reduces mature IL-2 production, thereby hampering murine T cell activation. Notably, the distinct expression kinetics of the Egr1-Nab2-IL-2 pathway between mouse and human render human T cells refractory to this vulnerability. We propose that the rational temporal modulation of splicing or transcription during peak&nbsp;de novo&nbsp;expression of key effectors can be used to fine-tune stimulation-dependent biological outcomes. Our data also show that critical consideration is required when extrapolating mouse data to the human system in basic and translational research.

T cells are nodal players in the adaptive immune response against pathogens and malignant cells. Alternative splicing plays a crucial role in T cell activation, which is analyzed mainly at later time points upon stimulation. Here we have discovered a 2-h time window early after stimulation where optimal splicing efficiency or, more generally, gene expression efficiency is crucial for successful T cell activation. Reducing the splicing efficiency at 4 to 6 h poststimulation significantly impaired murine T cell activation, which was dependent on the expression dynamics of the Egr1-Nab2-interleukin-2 (IL-2) pathway. This time window overlaps the time of peak IL-2&nbsp;de novo&nbsp;transcription, which, we suggest, represents a permissive time window in which decreased splicing (or transcription) efficiency reduces mature IL-2 production, thereby hampering murine T cell activation. Notably, the distinct expression kinetics of the Egr1-Nab2-IL-2 pathway between mouse and human render human T cells refractory to this vulnerability. We propose that the rational temporal modulation of splicing or transcription during peak&nbsp;de novo&nbsp;expression of key effectors can be used to fine-tune stimulation-dependent biological outcomes. Our data also show that critical consideration is required when extrapolating mouse data to the human system in basic and translational research.

Read the full publication here: <a href="https://doi.org/10.1128/MCB.00035-19" target="_blank" rel="noreferrer noopener">https://doi.org/10.1128/MCB.00035-19</a>

Read the full publication here: <a href="https://doi.org/10.1128/MCB.00035-19" target="_blank" rel="noreferrer noopener">https://doi.org/10.1128/MCB.00035-19</a>

Omiqa author: Alexander Neumann

Omiqa author: Alexander Neumann

Differential Interleukin-2 Transcription Kinetics Render Mouse but Not Human T Cells Vulnerable to Splicing Inhibition Early after Activation

Published June 2019 in Nucleic Acids Research, in collaboration with Freie Universität Berlin.

Published June 2019 in Nucleic Acids Research, in collaboration with Freie Universität Berlin.

In the yeast U1 snRNP the Prp39/Prp42 heterodimer is essential for early steps of spliceosome assembly. In metazoans no Prp42 ortholog exists, raising the question how the heterodimer is functionally substituted. Here we present the crystal structure of murine PRPF39, which forms a homodimer. Structure-guided point mutations disrupt dimer formation and inhibit splicing, manifesting the homodimer as functional unit. PRPF39 expression is controlled by NMD-inducing alternative splicing in mice and human, suggesting a role in adapting splicing efficiency to cell type specific requirements. A phylogenetic analysis reveals coevolution of shortened U1 snRNA and the absence of Prp42, which correlates with overall splicing complexity in different fungi. While current models correlate the diversity of spliceosomal proteins with splicing complexity, our study highlights a contrary case. We find that organisms with higher splicing complexity have substituted the Prp39/Prp42 heterodimer with a PRPF39 homodimer.

In the yeast U1 snRNP the Prp39/Prp42 heterodimer is essential for early steps of spliceosome assembly. In metazoans no Prp42 ortholog exists, raising the question how the heterodimer is functionally substituted. Here we present the crystal structure of murine PRPF39, which forms a homodimer. Structure-guided point mutations disrupt dimer formation and inhibit splicing, manifesting the homodimer as functional unit. PRPF39 expression is controlled by NMD-inducing alternative splicing in mice and human, suggesting a role in adapting splicing efficiency to cell type specific requirements. A phylogenetic analysis reveals coevolution of shortened U1 snRNA and the absence of Prp42, which correlates with overall splicing complexity in different fungi. While current models correlate the diversity of spliceosomal proteins with splicing complexity, our study highlights a contrary case. We find that organisms with higher splicing complexity have substituted the Prp39/Prp42 heterodimer with a PRPF39 homodimer.

Read the full publication here: <a href="https://doi.org/10.1093/nar/gkz243" target="_blank" rel="noreferrer noopener">https://doi.org/10.1093/nar/gkz243</a>

Read the full publication here: <a href="https://doi.org/10.1093/nar/gkz243" target="_blank" rel="noreferrer noopener">https://doi.org/10.1093/nar/gkz243</a>

Increased versatility despite reduced molecular complexity: evolution, structure and function of metazoan splicing factor PRPF39

Published April 2019 in Journal of Cell Science, in collaboration with Freie Universität Berlin.

Published April 2019 in Journal of Cell Science, in collaboration with Freie Universität Berlin.