Ribonucleoproteins (RNPs) mediate key cellular functions such as gene expression and its regulation. Whereas most RNP enzymes are stable in composition and harbor preformed active sites, the spliceosome, which removes noncoding introns from precursor messenger RNAs (pre-mRNAs), follows fundamentally different strategies. In order to provide both accuracy to the recognition of reactive splice sites in the pre-mRNA and flexibility to the choice of splice sites during alternative splicing, the spliceosome exhibits exceptional compositional and structural dynamics that are exploited during substrate-dependent complex assembly, catalytic activation, and active site remodeling.

One of the most amazing findings in molecular biology was the discovery that eukaryotic genes are discontinuous, with coding DNA being interrupted by stretches of non-coding sequence. The subsequent realization that the intervening regions are removed from pre-mRNA transcripts via the activity of a common set of small nuclear RNAs (snRNAs), which assemble together with associated proteins into a complex known as the spliceosome, was equally surprising. How do cells coordinate the assembly of this molecular machine? And how does the spliceosome accurately recognize exons and introns to carry out the splicing reaction? Insights into these questions have been gained by studying the life cycle of spliceosomal snRNAs from their transcription, nuclear export and re-import to their dynamic assembly into the spliceosome. This assembly process can also affect the regulation of alternative splicing and has implications for human disease.

Messenger RNA (mRNA) 3′ end formation is a nuclear process through which all eukaryotic primary transcripts are endonucleolytically cleaved and most of them acquire a poly(A) tail. This process, which consists in the recognition of defined poly(A) signals of the pre-mRNAs by a large cleavage/polyadenylation machinery, plays a critical role in gene expression. Indeed, the poly(A) tail of a mature mRNA is essential for its functions, including stability, translocation to the cytoplasm and translation. In addition, this process serves as a bridge in the network connecting the different transcription, capping, splicing and export machineries. It also participates in the quantitative and qualitative regulation of gene expression in a variety of biological processes through the selection of single or alternative poly(A) signals in transcription units. A large number of protein factors associates with this machinery to regulate the efficiency and specificity of this process and to mediate its interaction with other nuclear events. Here, we review the eukaryotic 3′ end processing machineries as well as the comprehensive set of regulatory factors and discuss the different molecular mechanisms of 3′ end processing regulation by proposing several overlapping models of regulation.

AbstractAlternative RNA processing mechanisms, including alternative splicing and alternative polyadenylation, are increasingly recognized as important regulators of gene expression. This article will focus on what has recently been described about alternative polyadenylation in development, differentiation, and disease in higher eukaryotes. We will also describe how the evolving global methodologies for examining the cellular transcriptome, both experimental and bioinformatic, are revealing new details about the complex nature of alternative 3′ end formation as well as interactions with other RNA‐mediated and RNA processing mechanisms. WIREs RNA 2011 2 22–31 DOI: 10.1002/wrna.47This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein–RNA Recognition RNA Processing > 3' End Processing

ABSTRACT The eukaryotic translation initiation factor eIF4E acts as a multifunctional factor that simultaneously influences mRNA processing, export, and translation in many organisms. Its multifactorial effects are derived from its capacity to bind to the methyl-7-guanosine cap on the 5’end of mRNAs and thus can act as a cap chaperone for transcripts in the nucleus and cytoplasm. In this review, we describe the multifactorial roles of eIF4E in major mRNA-processing events including capping, splicing, cleavage and polyadenylation, nuclear export and translation. We discuss the evidence that eIF4E acts at two levels to generate widescale changes to processing, export and ultimately the protein produced. First, eIF4E alters the production of components of the mRNA processing machinery, supporting a widescale reprogramming of multiple mRNA processing events. In this way, eIF4E can modulate mRNA processing without physically interacting with target transcripts. Second, eIF4E also physically interacts with both capped mRNAs and components of the RNA processing or translation machineries. Further, specific mRNAs are sensitive to eIF4E only in particular mRNA processing events. This selectivity is governed by the presence of cis-acting elements within mRNAs known as USER codes that recruit relevant co-factors engaging the appropriate machinery. In all, we describe the molecular bases for eIF4E’s multifactorial function and relevant regulatory pathways, discuss the basis for selectivity, present a compendium of ~80 eIF4E-interacting factors which play roles in these activities and provide an overview of the relevance of its functions to its oncogenic potential. Finally, we summarize early-stage clinical studies targeting eIF4E in cancer.

In eukaryotes, the separation of transcription from translation enables extensive mRNA processing (capping, splicing, and polyadenylation) before translation. This review focuses on recent work that provides considerable insight into how mRNAs navigate these processes in which a spectrum of RNA-binding proteins (RBPs) coordinate different processing steps and couple them to nuclear export. Although the principal components in these pathways have been identified, the precise way in which RBPs bind to mRNAs, some aspects of how their binding and release are mediated by DEAD-box ATPases, and the complete structures of some messenger ribonucleoprotein complexes (mRNPs) remain unclear. Moreover, the checkpoints that recognize both completion of mRNA processing and the generation of mature mRNPs, as well as how they are coordinated, are only partially characterized.

Cellular stresses regulate transcriptional readthrough, whereby RNA polymerase II elongates past a gene's polyadenylation cleavage site without RNA cleavage. Readthrough has been reported in several cancer types. Here, we use long-read sequencing of nascent RNA to quantify transcriptional readthrough in chronic myeloid leukemia (CML) cells and characterize early responses to the targeted therapeutic, imatinib. We show that the amount, length, and gene specificity of readthrough increase within 1 hour, before gene expression and alternative splicing alterations emerge. Notably, imatinib-dependent messenger RNA (mRNA) isoform changes involved "readthrough chimeras," in which exons from an upstream gene are alternatively spliced to exons in a downstream gene. Altered mRNA isoforms and chimera levels were detected in imatinib-resistant K562 cells as well as cells of patients with CML. Thus, imatinib can provoke a cascade of early changes to transcription and splicing fidelity that may lead to longer-term adjustments in gene expression, cancer cell differentiation, and the development of therapy resistance.

mRNA maturation requires precise coordination among transcription, 5' capping, splicing, and 3' end formation. Recent biochemical, structural, and genomic studies demonstrate that these processes are tightly coupled through dynamic interactions among RNA polymerase II, the spliceosome, and cleavage-polyadenylation complexes. Here, we synthesize current mechanistic insights into how transcription elongation factors and RNA processing machineries communicate to ensure efficient and accurate transcript maturation. We propose a "U1 relay" model as a unified framework for understanding co-transcriptional splicing and 3' end formation. We further discuss how RNAs are sorted into nuclear retention/degradation or export pathways based on the RNA processing status. Importantly, RNA processing factors not only act downstream of transcription but also feed back to modulate transcriptional elongation, pausing, and termination, thereby reinforcing bidirectional coupling between RNA synthesis and processing.

Abstract Virtually, all eukaryotic mRNAs are synthesized as precursor molecules that need to be extensively processed in order to serve as a blueprint for proteins. The three most prevalent processing steps are the capping reaction at the 5′‐end, the removal of intervening sequences by splicing, and the formation of poly (A)‐tails at the 3′‐end of the message by polyadenylation. A large number of proteins and small nuclear ribonucleoprotein complexes (snRNPs) interact with the mRNA and enable the different maturation steps. This chapter focuses on the biogenesis of snRNPs, the major components of the pre‐mRNA splicing machinery (spliceosome). A large body of evidence has revealed an intricate and segmented pathway for the formation of snRNPs that involves nucleo‐cytoplasmic transport events and elaborates assembly strategies. We summarize the knowledge about the different steps with an emphasis on trans ‐acting factors of snRNP maturation of higher eukaryotes. WIREs RNA 2011 2 718–731 DOI: 10.1002/wrna.87 This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes RNA Processing > Splicing Mechanisms RNA Export and Localization > Nuclear Export/Import RNA in Disease and Development > RNA in Disease

In vitro 3' end processing and poly(A) tailing of RNA in Trypanosoma cruzi. Pre-mRNA in kinetoplastids is processed to maturity following unique pathways requiring a transplicing event that links a common 39 nucleotide leader to the 5' termini of the mature mRNAs. The mechanisms of this reaction and other steps of mRNA processing; i.e., 5' capping and 3' cleavage and polyadenylation, have not been resolved. Herein, we describe a 3' polyadenylation activity in cell-free extracts prepared from nuclei isolated from Trypanosoma cruzi, the kinetoplastid agent of Chagas' Disease. Synthetic RNA transcripts incubated in these extracts in the presence of ATP are 3' polyadenylated. This polyadenylation activity is sensitive to heat or pre-treatment of the extract with Micrococcal nuclease, suggesting that an RNA-protein complex is required. As these are characteristics of polyadenylation activities in other eukaryotes, we believe that this activity may participate in the in vivo trypanosome mRNA polyadenylation system.

mRNA or pre-mRNA. This pre-mRNA must undergo extensive processing (including 5' capping, splicing to remove non-coding introns, and 3' polyadenylation) Messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein. mRNA is created during the process of transcription, where an enzyme (RNA polymerase) converts the gene into primary transcript mRNA (also known as pre-mRNA). This pre-mRNA usually still contains introns, regi Transcription is the process by which genetic information stored in DNA is copied into RNA by the enzyme RNA polymerase. During transcription, RNA polymerase binds to a promoter sequence on the DNA and synthesizes a complementary RNA strand (mRNA) from the DNA template. This process differs between prokaryotes and eukaryotes. In prokaryotes, transcription occurs in the cytoplasm. Because prokaryotes lack a membrane-bound nucleus, ribosomes can attach to the nascent mRNA strand and begin translation while transcription is still in progress. In eukaryotes, transcription occurs within the cell nucleus. The initial product of transcription is not functional mRNA but is termed precursor mRNA or pre-mRNA. This pre-mRNA must undergo extensive processing (including 5' capping, splicing to remove non-coding introns, and 3' polyadenylation) to become mature mRNA. Once processed, the mature mRNA is exported from the nucleus to the cytoplasm for translation.

In eukaryotes, mRNA synthesis is catalyzed by RNA polymerase II and involves several distinct steps, including transcript initiation, elongation, cleavage, and transcript release. Splicing of RNA can occur during (co-transcriptional) or after (post-transcriptional) RNA synthesis. Thus, RNA synthesis and processing occurs through the concerted activity of dozens of enzymes, each of which is potentially susceptible to perturbation by small molecules. However, there are few, if any, high-throughput screening strategies for identifying drugs which perturb a specific step in RNA synthesis and processing. Here we have developed a high-throughput fluorescence microscopy approach in single cells to screen for inhibitors of specific enzymatic steps in RNA synthesis and processing. By utilizing the high affinity interaction between bacteriophage capsid proteins (MS2, PP7) and RNA stem loops, we are able to fluorescently label the intron and exon of a β-globin reporter gene in human cells. This approach allows one to measure the kinetics of transcription, splicing and release in both fixed and living cells using a tractable, genetically encoded assay in a stable cell line. We tested this reagent in a targeted screen of molecules that target chromatin readers and writers and identified three compounds that slow transcription elongation without changing transcription initiation.

Mitochondria play key roles in cellular energy metabolism in eukaryotes. Mitochondria of most organisms contain their own genome and specific transcription and translation machineries. The expression of angiosperm mtDNA involves extensive RNA-processing steps, such as RNA trimming, editing, and the splicing of numerous group II-type introns. Pentatricopeptide repeat (PPR) proteins are key players of plant organelle gene expression and RNA metabolism. In the present analysis, we reveal the function of the MITOCHONDRIAL SPLICING FACTOR 2 gene (MISF2, AT3G22670) and show that it encodes a mitochondria-localized PPR protein that is crucial for early embryo-development in Arabidopsis. Molecular characterization of embryo-rescued misf2 plantlets indicates that the splicing of nad2 intron 1 and thus respiratory complex I biogenesis are strongly compromised. Moreover, the molecular function seems conserved between MISF2 protein in Arabidopsis and its orthologous gene (EMP10) in maize, suggesting that the ancestor of MISF2/EMP10 was recruited to function in nad2 processing before the monocot-dicot divergence, ~200 million years ago. These data provide new insights into the function of nuclear-encoded factors in mitochondrial gene expression and respiratory chain biogenesis during plant embryo development.

pro mi Capping and polyadenylation 5 6 Gm L1234 Splicing |y]rr] - E intermediate Splicing eliminates … alternative splicing as long as we have known about splicing itself. Alternative splicing occurs … variable regions by mutation, gene splicing, and RNA splicing. The net result is that the organism

Summary: Removal of introns from pre-mRNAs is an essential step in eukaryotic gene expression, mediated by spliceosomes that contain snRNAs as key components. Although snRNAs are transcribed in the nucleus and function in the same compartment, all except U6 shuttle to the cytoplasm. Surprisingly, the physiological relevance for shuttling is unclear, in particular because the snRNAs in Saccharomyces cerevisiae were reported to remain nuclear. Here, we show that all yeast pre-snRNAs including U6 undergo a stepwise maturation process after nuclear export by Mex67 and Xpo1. Sm- and Lsm-ring attachment occurs in the cytoplasm and is important for the snRNA re-import, mediated by Cse1 and Mtr10. Finally, nuclear pre-snRNA cleavage and trimethylation of the 5′-cap finalizes shuttling. Importantly, preventing pre-snRNAs from being exported or processed results in faulty spliceosome assembly and subsequent genome-wide splicing defects. Thus, pre-snRNA export is obligatory for functional splicing and resembles an essential evolutionarily conserved quality assurance step. : Becker et al. show snRNA maturation in yeast involves nuclear export and re-import mediated by Mex67-Mtr2 and Xpo1/Crm1 and by Mtr10 and Cse1, respectively. They propose a model for obligatory shuttling in eukaryotes by showing spliceosomes assemble with immature snRNAs when export is prevented, resulting in defective spliceosomes and genome-wide splicing defects. Keywords: snRNA, splicing, mRNA, U6, Mex67, Xpo1/Crm1