Eukaryotic Splicing Research Articles

Insights into the role of N6-methyladenosine (m6A) in plant-virus interactions.

N6-methyladenosine (m6A) is a common and dynamic epitranscriptomic modification in eukaryotic RNAs, affecting stability, splicing, translation, and degradation. Recent technological advancements have revealed the complex nature of m6A modifications, highlighting their importance in plant and animal species. The m6A modification is a reversible process, with "writers" depositing methylation, "erasers" demethylating it, and "reader" proteins recognizing m6A and executing various biological functions. Studying the relationship between m6A methylation and viral infection is crucial. Animal viruses, including retroviruses, RNA viruses, and DNA viruses, often employ the host's m6A machinery to replicate or avoid immune responses. In plant viruses, host methyltransferases or demethylases can stabilize or degrade viral RNA, depending on the virus-host interaction. Additionally, viral infections can modify the host's m6A machinery, impacting the viral life cycle. This review examines the role of m6A modifications in plant viral pathogenesis, focussing on RNA viruses infecting crops like alfalfa, turnip, wheat, rice, and potato. Understanding the role of m6A in virus-host interactions can aid in studying plant viral disease development and discovering novel antiviral targets for crop protection. In this review, we summarize current information on m6A in RNA biology, focussing on its function in viral infections and plant-virus interactions.

COMPREHENSIVE MOLECULAR PROFILING OF UVEAL MELANOMA EVALUATED WITH GENE EXPRESSION PROFILING, PREFERENTIALLY EXPRESSED ANTIGEN IN MELANOMA EXPRESSION, AND NEXT-GENERATION SEQUENCING.

To determine the association between gene-expression profiling (GEP), next-generation sequencing (NGS), preferentially expressed antigen in melanoma (PRAME) features, and metastatic risk in patients with uveal melanoma (UM). A retrospective analysis of patients with UM treated by brachytherapy or enucleation by a single ocular oncologist was conducted from November 2020 and July 2022. Clinicopathologic features, patient outcomes, GEP classification, NGS, and PRAME results were recorded. Comprehensive GEP, PRAME, and NGS testing was performed on 135 UMs. The presence of eukaryotic translation initiation factor 1A, X-chromosomal and splicing factor 3B subunit 1 mutations was significantly associated with GEP class 1A and GEP class 1B, respectively. The presence of BRCA- associated protein-1 mutation was significantly associated with GEP class 2. The average largest basal diameter for tumors with eukaryotic translation initiation factor 1A, X-chromosomal mutations was significantly smaller than those with splicing factor 3B subunit 1 mutations and BRCA1-associated protein-1 mutations. Class 2 tumors metastasized sooner than GEP class 1 tumors. Tumors with splicing factor 3B subunit 1 and/or BRCA1-associated protein-1 mutations metastasized sooner compared with tumors that had either no driver mutation or no mutations at all. Tumors with splicing factor 3B subunit 1 did not have a significantly different time to metastasis compared with tumors with BRCA1-associated protein-1 (P value = 0.97). Forty tumors (30%) were PRAME positive, and the remaining 95 tumors (70%) were PRAME negative. Tumors with PRAME-positive status did not have a significantly different time to metastasis compared with tumors without PRAME-positive status (P value = 0.11). GEP, NGS, and PRAME expression analysis help determine different levels of metastatic risk in UM. Although other prognostic tests exist, the following study reports on the use of NGS for metastatic prognostication in UM. However, limitations of NGS exist, especially with small lesions that are technically difficult to biopsy.

Optimization of a DNA-launched SARS-CoV-2 replicon with RNA splicing inhibitor Isoginkgetin.

We have previously developed a bacterial artificial chromosome (BAC)-vectored SARS-CoV-2 replicon, namely BAC-CoV2-Rep, which, upon transfection into host cells, serves as a transcription template for SARS-CoV-2 replicon mRNA to initiate replicon replication and produce nanoluciferase (Nluc) reporter from the subgenomic viral mRNA. However, an inherent issue of such DNA-launched replicon system is that the nascent full-length replicon transcript undergoes process by host RNA splicing machinery, which reduces replicon replication and generates spliced mRNA species expressing NLuc reporter independent of replicon replication. To mitigate this problem, we employed Isoginkgetin, a universal eukaryotic host splicing inhibitor, to treat cells transfected with BAC-CoV2-Rep. Isoginkgetin effectively increased the level of full-length replicon transcripts while concurrently reducing the level of Nluc signal derived from spliced replicon mRNA, making the Nluc reporter signal more correlated with replicon replication, as evidenced by treatment with known SARS-CoV-2 replication inhibitors including Remdesivir, GC376, and EIDD-1931. Thus, our study emphasizes that host RNA splicing is a confounding factor for DNA-launched SARS-CoV-2 replicon systems, which can be mitigated by Isoginkgetin treatment.

Monovalent metal ion binding promotes the first transesterification reaction in the spliceosome

Cleavage and formation of phosphodiester bonds in nucleic acids is accomplished by large cellular machineries composed of both protein and RNA. Long thought to rely on a two-metal-ion mechanism for catalysis, structure comparisons revealed many contain highly spatially conserved second-shell monovalent cations, whose precise function remains elusive. A recent high-resolution structure of the spliceosome, essential for pre-mRNA splicing in eukaryotes, revealed a potassium ion in the active site. Here, we employ biased quantum mechanics/ molecular mechanics molecular dynamics to elucidate the function of this monovalent ion in splicing. We discover that the K+ ion regulates the kinetics and thermodynamics of the first splicing step by rigidifying the active site and stabilizing the substrate in the pre- and post-catalytic state via formation of key hydrogen bonds. Our work supports a direct role for the K+ ion during catalysis and provides a mechanistic hypothesis likely shared by other nucleic acid processing enzymes.

Spliceosomal introns in the diplomonad parasite Giardia duodenalis revisited.

Complete reference genomes, including correct feature annotations, are a fundamental aspect of genomic biology. In the case of protozoan species such as Giardia duodenalis, a major human and animal parasite worldwide, accurate genome annotation can deepen our understanding of the evolution of parasitism and pathogenicity by identifying genes underlying key traits and clinically relevant cellular mechanisms, and by extension, the development of improved prevention strategies and treatments. This study used bioinformatics analyses of Giardia mRNA libraries to characterize known introns and identify new intron candidates, working towards completion of the G. duodenalis assemblage A strain 'WB' genome and further elucidating Giardia's gene expression. By using a set of experimentally validated positive control loci to calibrate our intron detection pipeline, we were able to detect evidence of previously missed candidate splice junctions directly from expressed transcript data. These intron candidates were further studied in silico using NMDS (non-metric multidimensional scaling) clustering to determine shared characteristics and their relative importance such as secondary structure, splicing efficiency and motif conservation, and thus to refine intron models. Results from this study identified 34 new intron candidates, with several potential introns showing evidence that secondary structure of the mRNA molecule might play a more significant role in splicing than previously reported eukaryotic splicing activity mediated by a reduced spliceosome present in G. duodenalis.

Captured: the elusive eukaryotic tRNA splicing enzyme.

Captured: the elusive eukaryotic tRNA splicing enzyme.

Eukaryotic splicing machinery in the plant-virus battleground.

Plant virual infections are mainly caused by plant-virus parasitism which affects ecological communities. Some viruses are highly pathogen specific that can infect only specific plants, while some cancause widespread harm, such as tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV). After a virus infects the host, undergoes a series of harmful effects, including the destruction of host cell membrane receptors, changes in cell membrane components, cell fusion, and the production of neoantigens on the cell surface. Therefore, competition between the host and the virus arises. The virus starts gaining control of critical cellular functions of the host cells and ultimately affects the fate of the targetedhost plants. Among these critical cellular processes, alternative splicing (AS) is an essential posttranscriptional regulation process in RNA maturation, which amplify host protein diversity and manipulates transcript abundance in response to plant pathogens. AS is widespread in nearly all human genes and critical in regulating animal-virus interactions. In particular, an animal virus can hijack the host splicing machinery to re-organize its compartments for propagation. Changes in AS are known to cause human disease, and various AS events have been reported to regulate tissue specificity, development, tumour proliferation, and multi-functionality. However, the mechanisms underlying plant-virus interactions are poorly understood. Here, we summarize the current understanding of how viruses interact with their plant hosts compared with humans, analyze currently used and putative candidate agrochemicals to treat plant-viral infections, and finally discussed the potential research hotspots in the future. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing.

ASAS-EGB: A statistical framework for estimating allele-specific alternative splicing events using transcriptome data

Ubiquitous Alternative splicing (AS) in eukaryotes greatly increases the biodiversity of proteins and involves in disease and cancer. Allele-specific AS studies can facilitate the identification of cis-acting elements because both alleles share the same cellular environment. Due to the limited information provided on the exons defined by AS events, we propose a statistical framework and algorithm ASAS-EGB for ASAS analysis using the gene transcriptome. The framework obtains exclusively compatible sets of gene isoforms supporting each event isoform, and uses both phased and non-phased SNPs within the exons on the gene isoforms for inference. Using this strategy, we have demonstrated ASAS-EGB can yield better ASAS inferential performance than using event isoforms. ASAS-EGB supports both single-end and paired-end RNA-seq data, and we have proved its robustness using RNA-seq replicates of individual NA12878. ASAS-EGB builds Bayesian models for ASAS analysis, and the MCMC method is used to solve the problem. With more detailed annotations for individual genomes and transcriptomes appearing in the future, the algorithm proposed by the paper can provide better support for these data to reveal the regulatory mechanisms of individual genomes.

The evolution of pre-mRNA splicing and its machinery revealed by reduced extremophilic red algae.

The Cyanidiales are a group of mostly thermophilic and acidophilic red algae that thrive near volcanic vents. Despite their phylogenetic relationship, the reduced genomes of Cyanidioschyzon merolae and Galdieria sulphuraria are strikingly different with respect to pre-mRNA splicing, a ubiquitous eukaryotic feature. Introns are rare and spliceosomal machinery is extremely reduced in C.merolae, in contrast to G.sulphuraria. Previous studies also revealed divergent spliceosomes in the mesophilic red alga Porphyridium purpureum and the red algal derived plastid of Guillardia theta (Cryptophyta), along with unusually high levels of unspliced transcripts. To further examine the evolution of splicing in red algae, we compared C.merolae and G.sulphuraria, investigating splicing levels, intron position, intron sequence features, and the composition of the spliceosome. In addition to identifying 11 additional introns in C. merolae, our transcriptomic analysis also revealed typical eukaryotic splicing in G. sulphuraria, whereas most transcripts in C. merolae remain unspliced. The distribution of intron positions within their host genes was examined to provide insight into patterns of intron loss in red algae. We observed increasing variability of 5' splice sites and branch donor regions with increasing intron richness. We also found these relationships to be connected to reductions in and losses of corresponding parts of the spliceosome. Our findings highlight patterns of intron and spliceosome evolution in related red algae under the pressures of genome reduction.

Eukaryotic tRNA splicing- one goal, two strategies, many players.

Transfer RNAs (tRNAs) are transcribed as precursor molecules that undergo several maturation steps before becoming functional for protein synthesis. One such processing mechanism is the enzyme-catalysed splicing of intron-containing pre-tRNAs. Eukaryotic tRNA splicing is an essential process since intron-containing tRNAs cannot fulfil their canonical function at the ribosome. Splicing of pre-tRNAs occurs in two steps: The introns are first excised by a tRNA-splicing endonuclease and the exons are subsequently sealed by an RNA ligase. An intriguing complexity has emerged from newly identified tRNA splicing factors and their interplay with other RNA processing pathways during the past few years. This review summarises our current understanding of eukaryotic tRNA splicing and the underlying enzyme machinery. We highlight recent structural advances and how they have shaped our mechanistic understanding of tRNA splicing in eukaryotic cells. A special focus lies on biochemically distinct strategies for exon-exon ligation in fungi versus metazoans.

Jumper enables discontinuous transcript assembly in coronaviruses

Genes in SARS-CoV-2 and other viruses in the order of Nidovirales are expressed by a process of discontinuous transcription which is distinct from alternative splicing in eukaryotes and is mediated by the viral RNA-dependent RNA polymerase. Here, we introduce the DISCONTINUOUS TRANSCRIPT ASSEMBLYproblem of finding transcripts and their abundances given an alignment of paired-end short reads under a maximum likelihood model that accounts for varying transcript lengths. We show, using simulations, that our method, JUMPER, outperforms existing methods for classical transcript assembly. On short-read data of SARS-CoV-1, SARS-CoV-2 and MERS-CoV samples, we find that JUMPER not only identifies canonical transcripts that are part of the reference transcriptome, but also predicts expression of non-canonical transcripts that are supported by subsequent orthogonal analyses. Moreover, application of JUMPER on samples with and without treatment reveals viral drug response at the transcript level. As such, JUMPER enables detailed analyses of Nidovirales transcriptomes under varying conditions.

Interconnected Gene Networks Underpin the Clinical Overlap of HNRNPH1-Related and Rubinstein-Taybi Intellectual Disability Syndromes.

Interconnected Gene Networks Underpin the Clinical Overlap of HNRNPH1-Related and Rubinstein-Taybi Intellectual Disability Syndromes.

DEK domain-containing proteins control flowering time in Arabidopsis.

Evolutionarily conserved DEK domain-containing proteins have been implicated in multiple chromatin-related processes, mRNA splicing and transcriptional regulation in eukaryotes. Here, we show that two DEK proteins, DEK3 and DEK4, control the floral transition in Arabidopsis. DEK3 and DEK4 directly associate with chromatin of related flowering repressors, FLOWERING LOCUS C (FLC), and its two homologs, MADS AFFECTING FLOWERING4 (MAF4) and MAF5, to promote their expression. The binding of DEK3 and DEK4 to a histone octamer in vivo affects histone modifications at FLC, MAF4 and MAF5 loci. In addition, DEK3 and DEK4 interact with RNA polymerase II and promote the association of RNA polymerase II with FLC, MAF4 and MAF5 chromatin to promote their expression. Our results show that DEK3 and DEK4 directly interact with chromatin to facilitate the transcription of key flowering repressors and thus prevent precocious flowering in Arabidopsis.

Identification of Intronic Lariat-Derived Circular RNAs in Arabidopsis by RNA Deep Sequencing.

Lariat RNAs are well-known by-products of pre-mRNA splicing in eukaryotes, which are produced by the excised introns when the 5' splice site (5' ss) joins with the branchpoint (BP) during splicing. In general, most of lariat RNAs are usually linearized by RNA debranching enzyme 1 (DBR1), followed by degradation for intron turnover. However, with the high-throughput RNA sequencing technology and bioinformatics methods, increasing evidences have shown that many lariat RNAs can stably accumulate under physiological conditions in both animals and plants. Here, we describe a large-scale analysis to systematically identify the lariat RNAs (i.e., intronic circular RNAs) in Arabidopsis by utilizing the RNA-sequencing data.

Atomic-Level Mechanism of Pre-mRNA Splicing in Health and Disease.

Intron removal from premature-mRNA (pre-mRNA splicing) is an essential part of gene expression and regulation that is required for the production of mature, protein-coding mRNA. The spliceosome (SPL), a majestic machine composed of five small nuclear RNAs and hundreds of proteins, behaves as an eminent transcriptome tailor, efficiently performing splicing as a protein-directed metallo-ribozyme. To select and excise long and diverse intronic sequences with single-nucleotide precision, the SPL undergoes a continuous compositional and conformational remodeling, forming eight distinct complexes throughout each splicing cycle. Splicing fidelity is of paramount importance to preserve the integrity of the proteome. Mutations in splicing factors can severely compromise the accuracy of this machinery, leading to aberrant splicing and altered gene expression. Decades of biochemical and genetic studies have provided insights into the SPL's composition and function, but its complexity and plasticity have prevented an in-depth mechanistic understanding. Single-particle cryogenic electron microscopy techniques have ushered in a new era for comprehending eukaryotic gene regulation, providing several near-atomic resolution structures of the SPL from yeast and humans. Nevertheless, these structures represent isolated snapshots of the splicing process and are insufficient to exhaustively assess the function of each SPL component and to unravel particular facets of the splicing mechanism in a dynamic environment.In this Account, building upon our contributions in this field, we discuss the role of biomolecular simulations in uncovering the mechanistic intricacies of eukaryotic splicing in health and disease. Specifically, we showcase previous applications to illustrate the role of atomic-level simulations in elucidating the function of specific proteins involved in the architectural reorganization of the SPL along the splicing cycle. Moreover, molecular dynamics applications have uniquely contributed to decrypting the channels of communication required for critical functional transitions of the SPL assemblies. They have also shed light on the role of carcinogenic mutations in the faithful selection of key intronic regions and the molecular mechanism of splicing modulators. Additionally, we emphasize the role of quantum-classical molecular dynamics in unraveling the chemical details of pre-mRNA cleavage in the SPL and in its evolutionary ancestors, group II intron ribozymes. We discuss methodological pitfalls of multiscale calculations currently used to dissect the splicing mechanism, presenting future challenges in this field. The results highlight how atomic-level simulations can enrich the interpretation of experimental results. We envision that the synergy between computational and experimental approaches will aid in developing innovative therapeutic strategies and revolutionary gene modulation tools to fight the over 200 human diseases associated with splicing misregulation, including cancer and neurodegeneration.

A Conserved NRDE-2/MTR-4 Complex Mediates Nuclear RNAi in Caenorhabditis elegans.

Small regulatory RNAs, such as small interfering RNAs (siRNAs) and PIWI-interacting RNAs, regulate splicing, transcription, and genome integrity in many eukaryotes. In Caenorhabditis elegans, siRNAs bind nuclear Argonautes (AGOs), which interact with homologous premessenger RNAs to recruit downstream silencing effectors, such as NRDE-2, to direct cotranscriptional gene silencing [or nuclear RNA interference (RNAi)]. To further our understanding of the mechanism of nuclear RNAi, we conducted immunoprecipitation-mass spectrometry on C. elegans NRDE-2 The major NRDE-2 interacting protein identified was the RNA helicase MTR-4 Co-immunoprecipitation analyses confirmed a physical association between NRDE-2 and MTR-4 MTR-4 colocalizes with NRDE-2 within the nuclei of most/all C. elegans somatic and germline cells. MTR-4 is required for nuclear RNAi, and interestingly, MTR-4 is recruited to premessenger RNAs undergoing nuclear RNAi via a process requiring nuclear siRNAs, the nuclear AGO HRDE-1, and NRDE-2, indicating that MTR-4 is a component of the C. elegans nuclear RNAi machinery. Finally, we confirm previous reports showing that human (Hs)NRDE2 and HsMTR4 also physically interact. Our data show that the NRDE-2/MTR-4 interactions are evolutionarily conserved, and that, in C. elegans, the NRDE-2/MTR-4 complex contributes to siRNA-directed cotranscriptional gene silencing.

The integral spliceosomal component CWC15 is required for development in Arabidopsis

Efficient mRNA splicing is a prerequisite for protein biosynthesis and the eukaryotic splicing machinery is evolutionarily conserved among species of various phyla. At its catalytic core resides the activated splicing complex Bact consisting of the three small nuclear ribonucleoprotein complexes (snRNPs) U2, U5 and U6 and the so-called NineTeen complex (NTC) which is important for spliceosomal activation. CWC15 is an integral part of the NTC in humans and it is associated with the NTC in other species. Here we show the ubiquitous expression and developmental importance of the Arabidopsis ortholog of yeast CWC15. CWC15 associates with core components of the Arabidopsis NTC and its loss leads to inefficient splicing. Consistent with the central role of CWC15 in RNA splicing, cwc15 mutants are embryo lethal and additionally display strong defects in the female haploid phase. Interestingly, the haploid male gametophyte or pollen in Arabidopsis, on the other hand, can cope without functional CWC15, suggesting that developing pollen might be more tolerant to CWC15-mediated defects in splicing than either embryo or female gametophyte.

Cellular UAP56 interacts with the HBx protein of the hepatitis B virus and is involved in viral RNA nuclear export in hepatocytes

UAP56 is an essential factor in eukaryotic pre-mRNA splicing and mRNA export. Many viruses require cellular RNA export factors to efficiently export viral RNA. However, the mechanisms behind hepatitis B virus (HBV) RNA splicing and nuclear export remain poorly understood. Here, our data show that UAP56 interacts with the HBx protein. Moreover, we demonstrate that the Q-motif of UAP56, which regulates RNA-binding and helicase activity, is essential for the interaction of UAP56 with HBx. Both knockdown of UAP56 and deficiency of HBx impaired cytoplasmic accumulation of HBV RNA transcripts, whereas knockdown of UAP56 also reduced the level of HBV pregenomic RNA splicing variants. In addition, knockdown of Nxf1 induced HBV RNA nuclear accumulation. These findings provide unique insights into the mechanistic details of HBV RNA export and splicing.

Auxiliary domains of the HrpB bacterial DExH-box helicase shape its RNA preferences

ABSTRACTRNA helicases are fundamental players in RNA metabolism: they remodel RNA secondary structures and arrange ribonucleoprotein complexes. While DExH-box RNA helicases function in ribosome biogenesis and splicing in eukaryotes, information is scarce about bacterial homologs. HrpB is the only bacterial DExH-box protein whose structure is solved. Besides the catalytic core, HrpB possesses three accessory domains, conserved in all DExH-box helicases, plus a unique C-terminal extension (CTE). The function of these auxiliary domains remains unknown. Here, we characterize genetically and biochemically Pseudomonas aeruginosa HrpB homolog. We reveal that the auxiliary domains shape HrpB RNA preferences, affecting RNA species recognition and catalytic activity. We show that, among several types of RNAs, the single-stranded poly(A) and the highly structured MS2 RNA strongly stimulate HrpB ATPase activity. In addition, deleting the CTE affects only stimulation by structured RNAs like MS2 and rRNAs, while deletion of accessory domains results in gain of poly(U)-dependent activity. Finally, using hydrogen-deuterium exchange, we dissect the molecular details of HrpB interaction with poly(A) and MS2 RNAs. The catalytic core interacts with both RNAs, triggering a conformational change that reorients HrpB. Regions within the accessory domains and CTE are, instead, specifically responsive to MS2. Altogether, we demonstrate that in bacteria, like in eukaryotes, DExH-box helicase auxiliary domains are indispensable for RNA handling.

Retroelement origins of pre-mRNA splicing.

Recent cryo-EM structures of a group II intron caught in the process of invading DNA have given new insight into the mechanisms of both splicing and retrotransposition. Conformational dynamics involving the branch-site helix domain VI are responsible for substrate exchange between the two steps of splicing. These structural rearrangements have strong parallels with the movement of the branch-site helix in the spliceosome during catalysis. This is strong evidence for the spliceosome evolving from a group II intron ancestor. We observe other topological changes in the overall structure of the catalytic domain V that may occur in the spliceosome as well. Therefore, studying group II introns not only provides us with insight into the evolutionary origins of the spliceosome, but also may inform the design of experiments to further probe structure-function relationships in this eukaryotic splicing apparatus. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution.