RNA Processing Steps Research Articles

Physiological and pathological roles of the transcriptional kinases CDK12 and CDK13 in the central nervous system.

The cyclin-dependent kinases 12 (CDK12) and 13 (CDK13) govern several steps of gene expression, including transcription, RNA processing and translation. The main target of CDK12/13 is the serine 2 residue of the carboxy-terminal domain of RNA polymerase II (RNAPII), thus influencing the directionality, elongation rate and processivity of the enzyme. The CDK12/13-dependent regulation of RNAPII activity influences the expression of selected target genes with important functional roles in the proliferation and viability of all eukaryotic cells. Neuronal cells are particularly affected by the loss of CDK12/13, as result of the high dependency of neuronal genes on RNAPII processivity for their expression. Deregulation of CDK12/13 activity strongly affects brain physiology by influencingthe stemness potential and differentiation properties of neuronal precursor cells. Moreover, mounting evidence also suggest the involvement of CDK12/13 in brain tumours. Herein, we discuss the functional role(s) of CDK12 and CDK13 in gene expression regulation and highlight similarities and differences between these highly homologous kinases, with particular attention to their impact on brain physiology and pathology. Lastly, we provide an overview of CDK12/13 inhibitors and of their efficacy in brain tumours and other neoplastic diseases.

MBNL splicing factors regulate the microtranscriptome of skeletal muscles.

Muscleblind like splicing regulators (MBNLs) govern various RNA-processing steps, including alternative splicing, polyadenylation, RNA stabilityand mRNA intracellular localization. In myotonic dystrophy type 1 (DM1), the most common muscular dystrophy in adults, MBNLs are sequestered on toxic RNA containing expanded CUG repeats, which leads to disruption of MBNL-regulated processes and disease features of DM1. Herein, we show the significance of MBNLs in regulating microtranscriptome dynamics during the postnatal development of skeletal muscles and in microRNA (miRNA) misregulation observed in mouse models and patients with DM1. We identify multiple miRNAs sensitive to MBNL proteins insufficiency and reveal that many of them were postnatally regulated, which correlates with increases in the activity of these proteins during this process. In adult Mbnl1-knockout mice, miRNA expression exhibited an adult-to-newborn shift. We hypothesize that Mbnl1 deficiency influences miRNA levels through a combination of mechanisms. First, the absence of Mbnl1 protein results in alterations to the levels of pri-miRNAs. Second, MBNLs affect miRNA biogenesis by regulating the alternative splicing of miRNA primary transcripts. We propose that the expression of miR-23b, miR-27b and miR-24-1, produced from the same cluster, depends on the MBNL-sensitive inclusion of alternative exons containing miRNA sequences. Our findings suggest that MBNL sequestration in DM1 is partially responsible for altered miRNA activity. This study provides new insights into the biological roles and functions of MBNL proteins as regulators of miRNA expression in skeletal muscles.

Revealing the Arabidopsis AtGRP7 mRNA binding proteome by specific enhanced RNA interactome capture

BackgroundThe interaction of proteins with RNA in the cell is crucial to orchestrate all steps of RNA processing. RNA interactome capture (RIC) techniques have been implemented to catalogue RNA- binding proteins in the cell. In RIC, RNA-protein complexes are stabilized by UV crosslinking in vivo. Polyadenylated RNAs and associated proteins are pulled down from cell lysates using oligo(dT) beads and the RNA-binding proteome is identified by quantitative mass spectrometry. However, insights into the RNA-binding proteome of a single RNA that would yield mechanistic information on how RNA expression patterns are orchestrated, are scarce.ResultsHere, we explored RIC in Arabidopsis to identify proteins interacting with a single mRNA, using the circadian clock-regulated Arabidopsis thaliana GLYCINE-RICH RNA-BINDING PROTEIN 7 (AtGRP7) transcript, one of the most abundant transcripts in Arabidopsis, as a showcase. Seedlings were treated with UV light to covalently crosslink RNA and proteins. The AtGRP7 transcript was captured from cell lysates with antisense oligonucleotides directed against the 5’untranslated region (UTR). The efficiency of RNA capture was greatly improved by using locked nucleic acid (LNA)/DNA oligonucleotides, as done in the enhanced RIC protocol. Furthermore, performing a tandem capture with two rounds of pulldown with the 5’UTR oligonucleotide increased the yield. In total, we identified 356 proteins enriched relative to a pulldown from atgrp7 mutant plants. These were benchmarked against proteins pulled down from nuclear lysates by AtGRP7 in vitro transcripts immobilized on beads. Among the proteins validated by in vitro interaction we found the family of Acetylation Lowers Binding Affinity (ALBA) proteins. Interaction of ALBA4 with the AtGRP7 RNA was independently validated via individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP). The expression of the AtGRP7 transcript in an alba loss-of-function mutant was slightly changed compared to wild-type, demonstrating the functional relevance of the interaction.ConclusionWe adapted specific RNA interactome capture with LNA/DNA oligonucleotides for use in plants using AtGRP7 as a showcase. We anticipate that with further optimization and up scaling the protocol should be applicable for less abundant transcripts.

Structures of co-transcriptional RNA capping enzymes on paused transcription complex

The 5′-end capping of nascent pre-mRNA represents the initial step in RNA processing, with evidence demonstrating that guanosine addition and 2′-O-ribose methylation occur in tandem with early steps of transcription by RNA polymerase II, especially at the pausing stage. Here, we determine the cryo-EM structures of the paused elongation complex in complex with RNGTT, as well as the paused elongation complex in complex with RNGTT and CMTR1. Our findings show the simultaneous presence of RNGTT and the NELF complex bound to RNA polymerase II. The NELF complex exhibits two conformations, one of which shows a notable rearrangement of NELF-A/D compared to that of the paused elongation complex. Moreover, CMTR1 aligns adjacent to RNGTT on the RNA polymerase II stalk. Our structures indicate that RNGTT and CMTR1 directly bind the paused elongation complex, illuminating the mechanism by which 5’-end capping of pre-mRNA during transcriptional pausing.

The molecular machinery for maturation of primary mtDNA transcripts.

Human mitochondria harbour a circular, polyploid genome (mtDNA) encoding 11 messenger RNAs (mRNAs), two ribosomal RNAs (rRNAs) and 22 transfer RNAs (tRNAs). Mitochondrial transcription produces long, polycistronic transcripts that span almost the entire length of the genome, and hence contain all three types of RNAs. The primary transcripts then undergo a number of processing and maturation steps, which constitute key regulatory points of mitochondrial gene expression. The first step of mitochondrial RNA processing consists of the separation of primary transcripts into individual, functional RNA molecules and can occur by two distinct pathways. Both are carried out by dedicated molecular machineries that substantially differ from RNA processing enzymes found elsewhere. As a result, the underlying molecular mechanisms remain poorly understood. Over the last years, genetic, biochemical and structural studies have identified key players involved in both RNA processing pathways and provided the first insights into the underlying mechanisms. Here, we review our current understanding of RNA processing in mammalian mitochondria and provide an outlook on open questions in the field.

Co-transcriptional gene regulationin eukaryotes and prokaryotes.

Many steps of RNA processing occur during transcription by RNA polymerases. Co-transcriptional activities are deemed commonplace in prokaryotes, in which the lack of membrane barriers allows mixing of all gene expression steps, from transcription to translation. In the past decade, an extraordinary level of coordination between transcription and RNA processing has emerged in eukaryotes. In this Review, we discuss recent developments in our understanding of co-transcriptional gene regulation in both eukaryotes and prokaryotes, comparing methodologies and mechanisms, and highlight striking parallels in how RNA polymerases interact with the machineries that act on nascent RNA. The development of RNA sequencing and imaging techniques that detect transient transcription and RNA processing intermediates has facilitated discoveries of transcription coordination with splicing, 3'-end cleavage and dynamic RNA folding and revealed physical contacts between processing machineries and RNA polymerases. Such studies indicate that intron retention in a given nascent transcript can prevent 3'-end cleavage and cause transcriptional readthrough, which is a hallmark of eukaryotic cellular stress responses. We also discuss how coordination between nascentRNA biogenesis and transcription drives fundamental aspects of gene expression in both prokaryotes and eukaryotes.

Differences in N6-methyladenosine (m6A) methylation among the three major clonal lineages of Toxoplasma gondii tachyzoites

Toxoplasma gondii is an important zoonotic parasite which has over 200 genotypes worldwide. N6-methyladenosine (m6A) methylation is a common epigenetic modification in messenger RNAs (mRNAs), and has been implicated in many aspects of mRNA biology. However, little is known about the difference in m6A methylation among different genotypes of T. gondii. In the present study, we employed methylated RNA immunoprecipitation sequencing (MeRIP-seq) technology to identify key genes exhibiting m6A methylation in the three major clonal lineages (Types I, II and III) of T. gondii tachyzoites. A total of 7650, 8359 and 7264 m6A peaks were identified in 5211, 5607 and 4974 genes in tachyzoites of RH strain (Type I), ME49 strain (Type II) and VEG strain (Type III), respectively. By comparing RH vs. ME49, RH vs. VEG, and ME49 vs. VEG, 735, 192 and 615 differentially methylated peaks (DMPs) were identified in 676, 168 and 553 genes, respectively. A combined MeRIP-seq and RNA-seq analysis revealed 172, 41 and 153 differentially methylated genes (DMGs) at both the m6A methylation and transcriptional level. Gene ontology term enrichment analysis of the DMPs identified differences related to Golgi apparatus, plasma membrane, signal transduction, RNA processing and catalytic step 2 spliceosome. KEGG pathway enrichment analysis showed that the DMGs are mainly involved in endocytosis, systemic lupus erythematosus and mTOR signaling pathway. These findings reveal genotype-specific differences in m6A methylation, which provide new resources for further investigations of the role of m6A in the pathobiology of T. gondii.

Minor introns impact on hematopoietic malignancies

In the intricate orchestration of the central dogma, pre-mRNA splicing plays a crucial role in the post-transcriptional process that transforms DNA into mature mRNA. Widely acknowledged as a pivotal RNA processing step, it significantly influences gene expression and alters the functionality of gene product proteins. Although U2-dependent spliceosomes efficiently manage the removal of over 99% of introns, a distinct subset of essential genes undergo splicing with a different intron type, denoted as minor introns, using U12-dependent spliceosomes. Mutations in spliceosome component genes are now recognized as prevalent genetic abnormalities in cancer patients, especially those with hematologic malignancies. Despite the relative rarity of minor introns, genes containing them are evolutionarily conserved and play crucial roles in functions such as the RAS-MAPK pathway. Disruptions in U12-type minor intron splicing caused by mutations in snRNA or its regulatory components significantly contribute to cancer progression. Notably, recurrent mutations associated with myelodysplastic syndrome (MDS) in the minor spliceosome component ZRSR2 underscore its significance. Examination of ZRSR2-mutated MDS cells has revealed that only a subset of minor spliceosome-dependent genes, such as LZTR1, consistently exhibit missplicing. Recent technological advancements have uncovered insights into minor introns, raising inquiries beyond current understanding. This review comprehensively explores the importance of minor intron regulation, the molecular implications of minor (U12-type) spliceosomal mutations and cis-regulatory regions, and the evolutionary progress of studies on minor, aiming to provide a sophisticated understanding of their intricate role in cancer biology.

RNA-seq analysis reveals transcriptome reprogramming and alternative splicing during early response to salt stress in tomato root.

Salt stress is one of the dominant abiotic stress conditions that cause severe damage to plant growth and, in turn, limiting crop productivity. It is therefore crucial to understand the molecular mechanism underlying plant root responses to high salinity as such knowledge will aid in efforts to develop salt-tolerant crops. Alternative splicing (AS) of precursor RNA is one of the important RNA processing steps that regulate gene expression and proteome diversity, and, consequently, many physiological and biochemical processes in plants, including responses to abiotic stresses like salt stress. In the current study, we utilized high-throughput RNA-sequencing to analyze the changes in the transcriptome and characterize AS landscape during the early response of tomato root to salt stress. Under salt stress conditions, 10,588 genes were found to be differentially expressed, including those involved in hormone signaling transduction, amino acid metabolism, and cell cycle regulation. More than 700 transcription factors (TFs), including members of the MYB, bHLH, and WRKY families, potentially regulated tomato root response to salt stress. AS events were found to be greatly enhanced under salt stress, where exon skipping was the most prevalent event. There were 3709 genes identified as differentially alternatively spliced (DAS), the most prominent of which were serine/threonine protein kinase, pentatricopeptide repeat (PPR)-containing protein, E3 ubiquitin-protein ligase. More than 100 DEGs were implicated in splicing and spliceosome assembly, which may regulate salt-responsive AS events in tomato roots. This study uncovers the stimulation of AS during tomato root response to salt stress and provides a valuable resource of salt-responsive genes for future studies to improve tomato salt tolerance.

Regulation of alternative splicing: Functional interplay with epigenetic modifications and its implication to cancer.

Eukaryotic gene expression is intricately regulated at multiple levels. The protein-coding genes are first transcribed as pre-mRNAs in the nucleus and undergo a series of RNA processing steps before being transported into the cytoplasm for translation. During RNA processing, most human genes (>95%) undergo alternative splicing to generate multiple mRNA isoforms from a single gene, which effectively diversifies the genome complexity. Since the splicing of most genes occurs co-transcriptionally, the regulation layers of gene expression often show functional interactions with each other. In this review, we provide a brief overview of alternative splicing regulation in three different layers (controlled by the splicing machinery, transcription process, and chromatin structure), emphasizing the regulatory roles of epigenetic modifications and the crosstalk between these layers. Specifically, we categorize the major effects of the epigenetic modifications on alternative splicing into three different types: by affecting transcription rate, splicing factor recruitment, or the expression/activity of splicing factor. The dysregulation of epigenetics and splicing are extremely common in cancer, we also discuss the potential mechanisms of how epigenetic changes can lead to splicing dysregulation and their functional consequences. We aim to provide insights into the complicated regulation of different gene expression layers, which will shed light on the novel approaches to modulate disease-related splicing dysregulation. This article is categorized under: RNA Processing > 3' End Processing RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing RNA in Disease and Development > RNA in Disease.

Simultaneous multifunctional transcriptome engineering by CRISPR RNA scaffold.

RNA processing and metabolism are subjected to precise regulation in the cell to ensure integrity and functions of RNA. Though targeted RNA engineering has become feasible with the discovery and engineering of the CRISPR-Cas13 system, simultaneous modulation of different RNA processing steps remains unavailable. In addition, off-target events resulting from effectors fused with dCas13 limit its application. Here we developed a novel platform, Combinatorial RNA Engineering via Scaffold Tagged gRNA (CREST), which can simultaneously execute multiple RNA modulation functions on different RNA targets. In CREST, RNA scaffolds are appended to the 3' end of Cas13 gRNA and their cognate RNA binding proteins are fused with enzymatic domains for manipulation. Taking RNA alternative splicing, A-to-G and C-to-U base editing as examples, we developed bifunctional and tri-functional CREST systems for simultaneously RNA manipulation. Furthermore, by fusing two split fragments of the deaminase domain of ADAR2 to dCas13 and/or PUFc respectively, we reconstituted its enzyme activity at target sites. This split design can reduce nearly 99% of off-target events otherwise induced by a full-length effector. The flexibility of the CREST framework will enrich the transcriptome engineering toolbox for the study of RNA biology.

SERRATE: a key factor in coordinated RNA processing in plants.

The SERRATE (SE) protein is involved in the processing of RNA polymerase II (RNAPII) transcripts. It is associated with different complexes engaged in different aspects of plant RNA metabolism, including assemblies involved in transcription, splicing, polyadenylation, miRNA biogenesis, and RNA degradation. SE stability and interactome properties can be influenced by phosphorylation. SE exhibits an intriguing liquid-liquid phase separation property that may be important in the assembly of different RNA-processing bodies. Therefore, we propose that SE seems to participate in the coordination of different RNA-processing steps and can direct the fate of transcripts, targeting them for processing or degradation when they cannot be properly processed or are synthesized in excess.

Alternative Splicing Outcomes Across an RNA-Binding Protein Concentration Gradient

Alternative splicing (AS) is a dynamic RNA processing step that produces multiple RNA isoforms from a single pre-mRNA transcript and contributes to the complexity of the cellular transcriptome and proteome. This process is regulated through a network of cis-regulatory sequence elements and trans-acting factors, most-notably RNA binding proteins (RBPs). The muscleblind-like (MBNL) and RNA binding fox-1 homolog (RBFOX) are two well characterized families of RBPs that regulate fetal to adult AS transitions critical for proper muscle, heart, and central nervous system development. To better understand how the concentration of these RBPs influences AS transcriptome wide, we engineered a MBNL1 and RBFOX1 inducible HEK-293 cell line. Modest induction of exogenous RBFOX1 in this cell line modulated MBNL1-dependent AS outcomes in 3 skipped exon events, despite significant levels of endogenous RBFOX1 and RBFOX2. Due to background RBFOX levels, we conducted a focused analysis of dose-dependent MBNL1 skipped exon AS outcomes and generated transcriptome wide dose–response curves. Analysis of this data demonstrates that MBNL1-regulated exclusion events may require higher concentrations of MBNL1 protein to properly regulate AS outcomes compared to inclusion events and that multiple arrangements of YGCY motifs can produce similar splicing outcomes. These results suggest that rather than a simple relationship between the organization of RBP binding sites and a specific splicing outcome, that complex interaction networks govern both AS inclusion and exclusion events across a RBP gradient.

Regulation of the epigenome through RNA modifications.

Chemical modifications of nucleotides expand the complexity and functional properties of genomes and transcriptomes. A handful of modifications in DNA bases are part of the epigenome, wherein DNA methylation regulates chromatin structure, transcription, and co-transcriptional RNA processing. In contrast, more than 150 chemical modifications of RNA constitute the epitranscriptome. Ribonucleoside modifications comprise a diverse repertoire of chemical groups, including methylation, acetylation, deamination, isomerization, and oxidation. Such RNA modifications regulate all steps of RNA metabolism, including folding, processing, stability, transport, translation, and RNA's intermolecular interactions. Initially thought to influence all aspects of the post-transcriptional regulation of gene expression exclusively, recent findings uncovered a crosstalk between the epitranscriptome and the epigenome. In other words, RNA modifications feedback to the epigenome to transcriptionally regulate gene expression. The epitranscriptome achieves this feat by directly or indirectly affecting chromatin structure and nuclear organization. This review highlights how chemical modifications in chromatin-associated RNAs (caRNAs) and messenger RNAs (mRNAs) encoding factors involved in transcription, chromatin structure, histone modifications, and nuclear organization affect gene expression transcriptionally.

Predictive model of transcriptional elongation control identifies trans regulatory factors from chromatin signatures.

Promoter-proximal Polymerase II (Pol II) pausing is a key rate-limiting step for gene expression. DNA and RNA-binding trans-acting factors regulating the extent of pausing have been identified. However, we lack a quantitative model of how interactions of these factors determine pausing, therefore the relative importance of implicated factors is unknown. Moreover, previously unknown regulators might exist. Here we address this gap with a machine learning model that accurately predicts the extent of promoter-proximal Pol II pausing from large-scale genome and transcriptome binding maps and gene annotation and sequence composition features. We demonstrate high accuracy and generalizability of the model by validation on an independent cell line which reveals the model's cell line agnostic character. Model interpretation in light of prior knowledge about molecular functions of regulatory factors confirms the interconnection of pausing with other RNA processing steps. Harnessing underlying feature contributions, we assess the relative importance of each factor, quantify their predictive effects and systematically identify previously unknown regulators of pausing. We additionally identify 16 previously unknown 7SK ncRNA interacting RNA-binding proteins predictive of pausing. Our work provides a framework to further our understanding of the regulation of the critical early steps in transcriptional elongation.

IARA: a complete and curated atlas of the biogenesis of spliceosome machinery during RNA splicing.

Splicing is one of the most important post-transcriptional processing systems and is responsible for the generation of transcriptome diversity in all living eukaryotes. Splicing is regulated by the spliceosome machinery, which is responsible for each step of primary RNA processing. However, current molecules and stages involved in RNA splicing are still spread over different studies. Thus, a curated atlas of spliceosome-related molecules and all involved stages during RNA processing can provide all researchers with a reliable resource to better investigate this important mechanism. Here, we present IARA (website access: https://pucpr-bioinformatics.github.io/atlas/), an extensively curated and constantly updated catalog of molecules involved in spliceosome machinery. IARA has a map of the steps involved in the human splicing mechanism, and it allows a detailed overview of the molecules involved throughout the distinct steps of splicing.

Sequential Polyadenylation to Enable Alternative mRNA 3' End Formation.

In eukaryotic cells, a key RNA processing step to generate mature mRNA is the coupled reaction for cleavage and polyadenylation (CPA) at the 3' end of individual transcripts. Many transcripts are alternatively polyadenylated (APA) to produce mRNAs with different 3' ends that may either alter protein coding sequence (CDS-APA) or create different lengths of 3'UTR (tandem-APA). As the CPA reaction is intimately associated with transcriptional termination, it has been widely assumed that APA is regulated cotranscriptionally. Isoforms terminated at different regions may have distinct RNA stability under different conditions, thus altering the ratio of APA isoforms. Such differential impacts on different isoforms have been considered as post-transcriptional APA, but strictly speaking, this can only be considered "apparent" APA, as the choice is not made during the CPA reaction. Interestingly, a recent study reveals sequential APA as a new mechanism for post-transcriptional APA. This minireview will focus on this new mechanism to provide insights into various documented regulatory paradigms.

Characterization of the protein expression and localization of hnRNP family members during murine spermatogenesis.

Mammalian testis exhibits remarkably high transcriptome complexity, and spermatogenesis undergoes two periods of transcriptional cessation. These make the RNA-binding proteins (RBPs) the utmost importance during male germ cell development. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a large family of RBPs implicated in many steps of RNA processing; however, their roles in spermatogenesis are largely unknown. Here, we investigated the expression pattern of 12 hnRNP family members in mouse testes and found that most detected members are highly expressed in the testis. Furthermore, we found that most of the detected hnRNP proteins (hnRNPD, hnRNPK, hnRNPQ, hnRNPU, and hnRNPUL1) display the highest signals in the nuclei of pachytene spermatocytes, round spermatids, and Sertoli cells, whereas hnRNPE1 exclusively concentrates in the manchette of elongating spermatids. The expression of these hnRNP proteins showed both similarities and specificity, suggesting their diverse roles in spermatogenesis.

DEAD-ly Affairs: The Roles of DEAD-Box Proteins on HIV-1 Viral RNA Metabolism.

In order to ensure viral gene expression, Human Immunodeficiency virus type-1 (HIV-1) recruits numerous host proteins that promote optimal RNA metabolism of the HIV-1 viral RNAs (vRNAs), such as the proteins of the DEAD-box family. The DEAD-box family of RNA helicases regulates multiple steps of RNA metabolism and processing, including transcription, splicing, nucleocytoplasmic export, trafficking, translation and turnover, mediated by their ATP-dependent RNA unwinding ability. In this review, we provide an overview of the functions and role of all DEAD-box family protein members thus far described to influence various aspects of HIV-1 vRNA metabolism. We describe the molecular mechanisms by which HIV-1 hijacks these host proteins to promote its gene expression and we discuss the implications of these interactions during viral infection, their possible roles in the maintenance of viral latency and in inducing cell death. We also speculate on the emerging potential of pharmacological inhibitors of DEAD-box proteins as novel therapeutics to control the HIV-1 pandemic.

40S hnRNP particles are a novel class of nuclear biomolecular condensates.

Heterogenous nuclear ribonucleoproteins (hnRNPs) are abundant proteins implicated in various steps of RNA processing that assemble on nuclear RNA into larger complexes termed 40S hnRNP particles. Despite their initial discovery 55 years ago, our understanding of these intriguing macromolecular assemblies remains limited. Here, we report the biochemical purification of native 40S hnRNP particles and the determination of their complete protein composition by label-free quantitative mass spectrometry, identifying A-group and C-group hnRNPs as the major protein constituents. Isolated 40S hnRNP particles dissociate upon RNA digestion and can be reconstituted in vitro on defined RNAs in the presence of the individual protein components, demonstrating a scaffolding role for RNA in nucleating particle formation. Finally, we revealed their nanometer scale, condensate-like nature, promoted by intrinsically disordered regions of A-group hnRNPs. Collectively, we identify nuclear 40S hnRNP particles as novel dynamic biomolecular condensates.