Archaeal Kink-Turn Binding Protein Mediates Inhibition of Orthomyxovirus Splicing Biology.

Despite lacking a DNA intermediate, orthomyxoviruses complete their replication cycle in the nucleus and generate multiple transcripts by usurping the host splicing machinery. This biology results in dynamic changes of relative viral transcripts over time and dictates the replicative phase of the infection. Here, we demonstrate that the family of archaeal L7Ae proteins uniquely inhibit the splicing biology of influenza A virus, influenza B virus, and Salmon isavirus, revealing a common strategy utilized by Orthomyxoviridae members to achieve this dynamic. L7Ae-mediated inhibition of virus biology was lost with the generation of a splicing-independent strain of influenza A virus and attempts to select for an escape mutant resulted in variants that conformed to host splicing biology at significant cost to their overall fitness. As L7Ae recognizes conventional kink turns in various RNAs, these data implicate the formation of a similar structure as a shared strategy adopted by this virus family to coordinate their replication cycle. IMPORTANCE Here, we demonstrate that a family of proteins from archaea specifically inhibit this splicing biology of all tested members of the Orthomyxoviridae family. We show that this inhibition extends to influenza A virus, influenza B virus, and isavirus genera, while having no significant impact on the mammalian transcriptome or proteome. Attempts to generate an escape mutant against L7Ae-mediated inhibition resulted in mutations surrounding the viral splice sites and a significant loss of viral fitness. Together, these findings reveal a unique biology shared among diverse members of the Orthomyxoviridae family that may serve as a means to generate future universal therapeutics.

What connects splicing of transfer RNA precursor molecules with pontocerebellar hypoplasia?

Transfer RNAs (tRNAs) represent the most abundant class of RNA molecules in the cell and are key players during protein synthesis and cellular homeostasis. Aberrations in the extensive tRNA biogenesis pathways lead to severe neurological disorders in humans. Mutations in the tRNA splicing endonuclease (TSEN) and its associated RNA kinase cleavage factor polyribonucleotide kinase subunit 1 (CLP1) cause pontocerebellar hypoplasia (PCH), a heterogeneous group of neurodegenerative disorders, that manifest as underdevelopment of specific brain regions typically accompanied by microcephaly, profound motor impairments, and child mortality. Recently, we demonstrated that mutations leading to specific PCH subtypes destabilize TSEN in vitro and cause imbalances of immature to mature tRNA ratios in patient-derived cells. However, how tRNA processing defects translate to disease on a systems level has not been understood. Recent findings suggested that other cellular processes may be affected by mutations in TSEN/CLP1 and obscure the molecular mechanisms of PCH emergence. Here, we review PCH disease models linked to the TSEN/CLP1 machinery and discuss future directions to study neuropathogenesis.

Galectin-3-U1 snRNP Complexes Initiate Splicing Activity in U1-Depleted Nuclear Extracts.

Fractionation of HeLa cell nuclear extracts by glycerol gradient centrifugation separates endogenous uracil-rich small nuclear ribonucleoprotein complexes (U snRNP) into numerous particles sedimenting from 7S to greater than 60S. Complexes sedimenting at 10S contain a single U snRNP (U1 snRNP) and galectin-3. Addition of antibodies specific for galectin-3 to fractions containing these 10S complexes coprecipitates U1 snRNP, indicating that a fraction of the U1 snRNP is associated with this galectin. Galectin-3 has been shown by depletion-reconstitution studies to be an integral splicing component involved both in spliceosome assembly and splicing activity. The first step in initiation of spliceosome assembly is binding of U1 snRNP to the 5' splice site of the premessenger RNA substrate. The finding that U1 snRNP and galectin-3 are associated in splicing extracts hints that this complex affords a potential entry point for galectin-3 into the splicing pathway. Addition of U1 snRNP-galectin-3 complexes immunoselected from the 10S region of glycerol gradients to a U1-depleted nuclear extract initiates splicing activity with the formation of splicing intermediates and mature mRNA. This chapter describes the materials and methods for these experiments that document galectin-3-U1 snRNP complexes initiate the splicing reaction in a U1-depleted nuclear extract.

Recursive splicing is a rare event in the mouse brain.

Recursive splicing (RS) is a splicing mechanism to remove long introns from messenger RNA precursors of long genes. Compared to the hundreds of RS events identified in humans and drosophila, only ten RS events have been reported in mice. To further investigate RS in mice, we analyzed RS in the mouse brain, a tissue that is enriched in the expression of long genes. We found that nuclear total RNA sequencing is an efficient approach to investigate RS events. We analyzed 1.15 billion uniquely mapped reads from the nuclear total RNA sequencing data in the mouse cerebral cortex. Unexpectedly, we only identified 20 RS sites, suggesting that RS is a rare event in the mouse brain. We also identified that RS is constitutive between excitatory and inhibitory neurons and between sexes in the mouse cerebral cortex. In addition, we found that the primary sequence context is associated with RS splicing intermediates and distinguishes RS AGGT site from non-RS AGGT sites, indicating the importance of the primary sequence context in RS sites. Moreover, we discovered that cryptic exons may use an RS-like mechanism for splicing. Overall, we provide novel findings about RS in long genes in the mouse brain.

The DROL1 subunit of U5 snRNP in the spliceosome is specifically required to splice AT-AC-type introns in Arabidopsis.

An Arabidopsis mutant named defective repression of OLE3::LUC 1 (drol1) was originally isolated as a mutant with defects in the repression of OLEOSIN3 (OLE3) after seed germination. In this study, we show that DROL1 is an Arabidopsis homolog of yeast DIB1, a subunit of the U5 small nuclear ribonucleoprotein particle (snRNP) in the spliceosome. It is also part of a new subfamily that is specific to a certain class of eukaryotes. Comprehensive analysis of the intron splicing using RNA sequencing analysis of the drol1 mutants revealed that most of the minor introns with AT-AC dinucleotide termini had reduced levels of splicing. Only two nucleotide substitutions from AT-AC to GT-AG enabled AT-AC-type introns to be spliced in drol1 mutants. Forty-eight genes, including those having important roles in abiotic stress responses and cell proliferation, exhibited reduced splicing of AT-AC-type introns in the drol1 mutants. Additionally, drol1 mutant seedlings showed retarded growth, similar to that caused by the activation of abscisic acid signaling, possibly as a result of reduced AT-AC-type intron splicing in the endosomal Na+ /H+ antiporters and plant-specific histone deacetylases. These results indicate that DROL1 is specifically involved in the splicing of minor introns with AT-AC termini and that this plays an important role in plant growth and development.

Distinct roles of hnRNPH1 low-complexity domains in splicing and transcription.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins that control key events in RNA biogenesis under both normal and diseased cellular conditions. The low-complexity (LC) domain of hnRNPs can become liquid-like droplets or reversible amyloid-like polymers by phase separation. Yet, whether phase separation of the LC domains contributes to physiological functions of hnRNPs remains unclear. hnRNPH1 contains two LC domains, LC1 and LC2. Here, we show that reversible phase separation of the LC1 domain is critical for both interaction with different kinds of RNA-binding proteins and control of the alternative-splicing activity of hnRNPH1. Interestingly, although not required for phase separation, the LC2 domain contributes to the robust transcriptional activation of hnRNPH1 when fused to the DNA-binding domain, as found recently in acute lymphoblastic leukemia. Our data suggest that the ability of the LC1 domain to phase-separate into reversible polymers or liquid-like droplets is essential for function of hnRNPH1 as an alternative RNA-splicing regulator, whereas the LC2 domain may contribute to the aberrant transcriptional activity responsible for cancer transformation.

DHX15-independent roles for TFIP11 in U6 snRNA modification, U4/U6.U5 tri-snRNP assembly and pre-mRNA splicing fidelity.

The U6 snRNA, the core catalytic component of the spliceosome, is extensively modified post-transcriptionally, with 2'-O-methylation being most common. However, how U6 2'-O-methylation is regulated remains largely unknown. Here we report that TFIP11, the human homolog of the yeast spliceosome disassembly factor Ntr1, localizes to nucleoli and Cajal Bodies and is essential for the 2'-O-methylation of U6. Mechanistically, we demonstrate that TFIP11 knockdown reduces the association of U6 snRNA with fibrillarin and associated snoRNAs, therefore altering U6 2'-O-methylation. We show U6 snRNA hypomethylation is associated with changes in assembly of the U4/U6.U5 tri-snRNP leading to defects in spliceosome assembly and alterations in splicing fidelity. Strikingly, this function of TFIP11 is independent of the RNA helicase DHX15, its known partner in yeast. In sum, our study demonstrates an unrecognized function for TFIP11 in U6 snRNP modification and U4/U6.U5 tri-snRNP assembly, identifying TFIP11 as a critical spliceosome assembly regulator.

Structural basis of the interaction between SETD2 methyltransferase and hnRNP L paralogs for governing co-transcriptional splicing.

The RNA recognition motif (RRM) binds to nucleic acids as well as proteins. More than one such domain is found in the pre-mRNA processing hnRNP proteins. While the mode of RNA recognition by RRMs is known, the molecular basis of their protein interaction remains obscure. Here we describe the mode of interaction between hnRNP L and LL with the methyltransferase SETD2. We demonstrate that for the interaction to occur, a leucine pair within a highly conserved stretch of SETD2 insert their side chains in hydrophobic pockets formed by hnRNP L RRM2. Notably, the structure also highlights that RRM2 can form a ternary complex with SETD2 and RNA. Remarkably, mutating the leucine pair in SETD2 also results in its reduced interaction with other hnRNPs. Importantly, the similarity that the mode of SETD2-hnRNP L interaction shares with other related protein-protein interactions reveals a conserved design by which splicing regulators interact with one another.

HnRNP K mislocalisation is a novel protein pathology of frontotemporal lobar degeneration and ageing and leads to cryptic splicing.

Heterogeneous nuclear ribonucleoproteins (HnRNPs) are a group of ubiquitously expressed RNA-binding proteins implicated in the regulation of all aspects of nucleic acid metabolism. HnRNP K is a member of this highly versatile hnRNP family. Pathological redistribution of hnRNP K to the cytoplasm has been linked to the pathogenesis of several malignancies but, until now, has been underexplored in the context of neurodegenerative disease. Here we show hnRNP K mislocalisation in pyramidal neurons of the frontal cortex to be a novel neuropathological feature that is associated with both frontotemporal lobar degeneration and ageing. HnRNP K mislocalisation is mutually exclusive to TDP-43 and tau pathological inclusions in neurons and was not observed to colocalise with mitochondrial, autophagosomal or stress granule markers. De-repression of cryptic exons in RNA targets following TDP-43 nuclear depletion is an emerging mechanism of potential neurotoxicity in frontotemporal lobar degeneration and the mechanistically overlapping disorder amyotrophic lateral sclerosis. We silenced hnRNP K in neuronal cells to identify the transcriptomic consequences of hnRNP K nuclear depletion. Intriguingly, by performing RNA-seq analysis we find that depletion of hnRNP K induces 101 novel cryptic exon events. We validated cryptic exon inclusion in an SH-SY5Y hnRNP K knockdown and in FTLD brain exhibiting hnRNP K nuclear depletion. We, therefore, present evidence for hnRNP K mislocalisation to be associated with FTLD and for this to induce widespread changes in splicing.

Dynamic imaging of nascent RNA reveals general principles of transcription dynamics and stochastic splice site selection.

The activities of RNA polymerase and the spliceosome are responsible for the heterogeneity in the abundance and isoform composition of mRNA in human cells. However, the dynamics of these megadalton enzymatic complexes working in concert on endogenous genes have not been described. Here, we establish a quasi-genome-scale platform for observing synthesis and processing kinetics of single nascent RNA molecules in real time. We find that all observed genes show transcriptional bursting. We also observe large kinetic variation in intron removal for single introns in single cells, which is inconsistent with deterministic splice site selection. Transcriptome-wide footprinting of the U2AF complex, nascent RNA profiling, long-read sequencing, and lariat sequencing further reveal widespread stochastic recursive splicing within introns. We propose and validate a unified theoretical model to explain the general features of transcription and pervasive stochastic splice site selection.

Different roles for the adjoining and structurally similar A-rich and poly(A) domains of oskar mRNA: Only the A-rich domain is required for oskar noncoding RNA function, which includes MTOC positioning.

Drosophila oskar (osk) mRNA has both coding and noncoding functions, with the latter required for progression through oogenesis. Noncoding activity is mediated by the osk 3' UTR. Three types of cis elements act most directly and are clustered within the final ~120 nucleotides of the 3' UTR: multiple binding sites for the Bru1 protein, a short highly conserved region, and A-rich sequences abutting the poly(A) tail. Here we extend the characterization of these elements and their functions, providing new insights into osk noncoding RNA function and the makeup of the cis elements. We show that all three elements are required for correct positioning of the microtubule organizing center (MTOC), a defect not previously reported for any osk mutant. Normally, the MTOC is located at the posterior of the oocyte during previtellogenic stages of oogenesis, and this distribution underlies the strong posterior enrichment of many mRNAs transported into the oocyte from the nurse cells. When osk noncoding function was disrupted the MTOC was dispersed in the oocyte and osk mRNA failed to be enriched at the posterior, although transport to the oocyte was not affected. A previous study did not detect loss of posterior enrichment for certain osk mutants lacking noncoding activity (Kanke et al., 2015). This discrepancy may be due to use of imaging aimed at monitoring transport to the oocyte rather than posterior enrichment. Involvement in MTOC positioning suggests that the osk noncoding function may act in conjunction with genes whose loss has similar effects, and that osk function may extend to other processes requiring those genes. Further characterization of the cis elements required for osk noncoding function included completion of saturation mutagenesis of the most highly conserved region, providing critical information for evaluating the possible contribution of candidate binding factors. The 3'-most cis element is a cluster of A-rich sequences, the ARS. The close juxtaposition and structural similarity of the ARS and poly(A) tail raised the possibility that they comprise an extended A-rich element required for osk noncoding function. We found that absence of the poly(A) tail did not mimic the effects of mutation of the ARS, causing neither arrest of oogenesis nor mispositioning of osk mRNA in previtellogenic stage oocytes. Thus, the ARS and the poly(A) tail are not interchangeable for osk noncoding RNA function, suggesting that the role of the ARS is not in recruitment of Poly(A) binding protein (PABP), the protein that binds the poly(A) tail. Furthermore, although PABP has been implicated in transport of osk mRNA from the nurse cells to the oocyte, mutation of the ARS in combination with loss of the poly(A) tail did not disrupt transport of osk mRNA into the oocyte. We conclude that PABP acts indirectly in osk mRNA transport, or is associated with osk mRNA independent of an A-rich binding site. Although the poly(A) tail was not required for osk mRNA transport into the oocyte, its absence was associated with a novel osk mRNA localization defect later in oogenesis, potentially revealing a previously unrecognized step in the localization process.

Emerging role of non-coding RNA in health and disease.

Human diseases have always been a significant turf of concern since the origin of mankind. It is cardinal to know the cause, treatment, and cure for every disease condition. With the advent and advancement in technology, the molecular arena at the microscopic level to study the mechanism, progression, and therapy is more rational and authentic pave than a macroscopic approach. Non-coding RNAs (ncRNAs) have now emerged as indispensable players in the diagnosis, development, and therapeutics of every abnormality concerning physiology, pathology, genetics, epigenetics, oncology, and developmental diseases. This is a comprehensive attempt to collate all the existing and proven strategies, techniques, mechanisms of genetic disorders including Silver Russell Syndrome, Fascio- scapula humeral muscular dystrophy, cardiovascular diseases (atherosclerosis, cardiac fibrosis, hypertension, etc.), neurodegenerative diseases (Spino-cerebral ataxia type 7, Spino-cerebral ataxia type 8, Spinal muscular atrophy, Opitz-Kaveggia syndrome, etc.) cancers (cervix, breast, lung cancer, etc.), and infectious diseases (viral) studied so far. This article encompasses discovery, biogenesis, classification, and evolutionary prospects of the existence of this junk RNA along with the integrated networks involving chromatin remodelling, dosage compensation, genome imprinting, splicing regulation, post-translational regulation and proteomics. In conclusion, all the major human diseases are discussed with a facilitated technology transfer, advancements, loopholes, and tentative future research prospects have also been proposed.

The oxidoreductase PYROXD1 uses NAD(P)+ as an antioxidant to sustain tRNA ligase activity in pre-tRNA splicing and unfolded protein response.

The tRNA ligase complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), and Xbp1-mRNA during the unfolded protein response (UPR). In aerobic conditions, a cysteine residue bound to two metal ions in its ancient, catalytic subunit RTCB could make the tRNA-LC susceptible to oxidative inactivation. Here, we confirm this hypothesis and reveal a co-evolutionary association between the tRNA-LC and PYROXD1, a conserved and essential oxidoreductase. We reveal that PYROXD1 preserves the activity of the mammalian tRNA-LC in pre-tRNA splicing and UPR. PYROXD1 binds the tRNA-LC in the presence of NAD(P)H and converts RTCB-bound NAD(P)H into NAD(P)+, a typical oxidative co-enzyme. However, NAD(P)+ here acts as an antioxidant and protects the tRNA-LC from oxidative inactivation, which is dependent on copper ions. Genetic variants of PYROXD1 that cause human myopathies only partially support tRNA-LC activity. Thus, we establish the tRNA-LC as an oxidation-sensitive metalloenzyme, safeguarded by the flavoprotein PYROXD1 through an unexpected redox mechanism.

Conserved long-range base pairings are associated with pre-mRNA processing of human genes.

The ability of nucleic acids to form double-stranded structures is essential for all living systems on Earth. Current knowledge on functional RNA structures is focused on locally-occurring base pairs. However, crosslinking and proximity ligation experiments demonstrated that long-range RNA structures are highly abundant. Here, we present the most complete to-date catalog of conserved complementary regions (PCCRs) in human protein-coding genes. PCCRs tend to occur within introns, suppress intervening exons, and obstruct cryptic and inactive splice sites. Double-stranded structure of PCCRs is supported by decreased icSHAPE nucleotide accessibility, high abundance of RNA editing sites, and frequent occurrence of forked eCLIP peaks. Introns with PCCRs show a distinct splicing pattern in response to RNAPII slowdown suggesting that splicing is widely affected by co-transcriptional RNA folding. The enrichment of 3'-ends within PCCRs raises the intriguing hypothesis that coupling between RNA folding and splicing could mediate co-transcriptional suppression of premature pre-mRNA cleavage and polyadenylation.

SRSF9 Regulates Cassette Exon Splicing of Caspase-2 by Interacting with Its Downstream Exon.

Alternative splicing (AS) is an important posttranscriptional regulatory process. Damaged or unnecessary cells need to be removed though apoptosis to maintain physiological processes. Caspase-2 pre-mRNA produces pro-apoptotic long mRNA and anti-apoptotic short mRNA isoforms through AS. How AS of Caspase-2 is regulated remains unclear. In the present study, we identified a novel regulatory protein SRSF9 for AS of Caspase-2 cassette exon 9. Knock-down (KD) of SRSF9 increased inclusion of cassette exon and on the other hand, overexpression of SRSF9 decreased inclusion of this exon. Deletion mutagenesis demonstrated that exon 9, parts of intron 9, exon 8 and exon 10 were not required for the role of SRSF9 in Caspase-2 AS. However, deletion and substitution mutation analysis revealed that AGGAG sequence located at exon 10 provided functional target for SRSF9. In addition, RNA-pulldown mediated immunoblotting analysis showed that SRSF9 interacted with this sequence. Gene ontology analysis of RNA-seq from SRSF9 KD cells demonstrates that SRSF9 could regulate AS of a subset of apoptosis related genes. Collectively, our results reveal a basis for regulation of Caspase-2 AS.

The Physcomitrella patens chromatin adaptor PpMRG1 interacts with H3K36me3 and regulates light-responsive alternative splicing.

Plants perceive dynamic light conditions and optimize their growth and development accordingly by regulating gene expression at multiple levels. Alternative splicing (AS), a widespread mechanism in eukaryotes that post-transcriptionally generates two or more messenger RNAs (mRNAs) from the same pre-mRNA, is rapidly controlled by light. However, a detailed mechanism of light-regulated AS is still not clear. In this study, we demonstrate that histone 3 lysine 36 trimethylation (H3K36me3) rapidly and differentially responds to light at specific gene loci with light-regulated intron retention (IR) of their transcripts in the moss Physcomitrella patens. However, the level of H3K36me3 following exposure to light is inversely related to that of IR events. Physcomitrella patens MORF-related gene 1 (PpMRG1), a chromatin adaptor, bound with higher affinity to H3K36me3 in light conditions than in darkness and was differentially targeted to gene loci showing light-responsive IR. Transcriptome analysis indicated that PpMRG1 functions in the regulation of light-mediated AS. Furthermore, PpMRG1 was also involved in red light-mediated phototropic responses. Our results suggest that light regulates histone methylation, which leads to alterations of AS patterns. The chromatin adaptor PpMRG1 potentially participates in light-mediated AS, revealing that chromatin-coupled regulation of pre-mRNA splicing is an important aspect of the plant's response to environmental changes.

Alternative RNA Splicing in Fatty Liver Disease.

Alternative RNA splicing is a process by which introns are removed and exons are assembled to construct different RNA transcript isoforms from a single pre-mRNA. Previous studies have demonstrated an association between dysregulation of RNA splicing and a number of clinical syndromes, but the generality to common disease has not been established. Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease affecting one-third of adults worldwide, increasing the risk of cirrhosis and hepatocellular carcinoma (HCC). In this review we focus on the change in alternative RNA splicing in fatty liver disease and the role for splicing regulation in disease progression.

Unstructural Biology of TRP Ion Channels: The Role of Intrinsically Disordered Regions in Channel Function and Regulation.

The first genuine high-resolution single particle cryo-electron microscopy structure of a membrane protein determined was a transient receptor potential (TRP) ion channel, TRPV1, in 2013. This methodical breakthrough opened up a whole new world for structural biology and ion channel aficionados alike. TRP channels capture the imagination due to the sheer endless number of tasks they carry out in all aspects of animal physiology. To date, structures of at least one representative member of each of the six mammalian TRP channel subfamilies as well as of a few non-mammalian families have been determined. These structures were instrumental for a better understanding of TRP channel function and regulation. However, all of the TRP channel structures solved so far are incomplete since they miss important information about highly flexible regions found mostly in the channel N- and C-termini. These intrinsically disordered regions (IDRs) can represent between a quarter to almost half of the entire protein sequence and act as important recruitment hubs for lipids and regulatory proteins. Here, we analyze the currently available TRP channel structures with regard to the extent of these "missing" regions and compare these findings to disorder predictions. We discuss select examples of intra- and intermolecular crosstalk of TRP channel IDRs with proteins and lipids as well as the effect of splicing and post-translational modifications, to illuminate their importance for channel function and to complement the prevalently discussed structural biology of these versatile and fascinating proteins with their equally relevant 'unstructural' biology.

Splicing regulation in brain and testis: common themes for highly specialized organs.

Expansion of the coding and regulatory capabilities of eukaryotic transcriptomes by alternative splicing represents one of the evolutionary forces underlying the increased structural complexity of metazoans. Brain and testes stand out as the organs that mostly exploit the potential of alternative splicing, thereby expressing the largest repertoire of splice variants. Herein, we will review organ-specific as well as common mechanisms underlying the high transcriptome complexity of these organs and discuss the impact exerted by this widespread alternative splicing regulation on the functionality and differentiation of brain and testicular cells.