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In eukaryotes, RNAs transcribed by RNA Pol II are modified at the 5′ end with a 7-methylguanosine (m 7 G) cap, which is recognized by the nuclear cap binding complex (CBC). The CBC plays multiple important roles in mRNA metabolism, including transcription, splicing, polyadenylation, and export. It promotes mRNA export through direct interaction with a key mRNA export factor, ALYREF, which in turn links the TRanscription and EXport (TREX) complex to the 5′ end of mRNA. However, the molecular mechanism for CBC-mediated recruitment of the mRNA export machinery is not well understood. Here, we present the first structure of the CBC in complex with an mRNA export factor, ALYREF. The cryo-EM structure of CBC-ALYREF reveals that the RRM domain of ALYREF makes direct contact with both the NCBP1 and NCBP2 subunits of the CBC. Comparing CBC-ALYREF with other cellular complexes containing CBC and/or ALYREF components provides insights into the coordinated events during mRNA transcription, splicing, and export.

The NS1 protein of influenza A virus is a major virulence factor that is essential for pathogenesis. NS1 functions to impair innate and adaptive immunity by inhibiting host signal transduction and gene expression, but its mechanisms of action remain to be fully elucidated. We show here that NS1 forms an inhibitory complex with NXF1/TAP, p15/NXT, Rae1/mrnp41, and E1B-AP5, which are key constituents of the mRNA export machinery that interact with both mRNAs and nucleoporins to direct mRNAs through the nuclear pore complex. Increased levels of NXF1, p15, or Rae1 revert the mRNA export blockage induced by NS1. Furthermore, influenza virus down-regulates Nup98, a nucleoporin that is a docking site for mRNA export factors. Reduced expression of these mRNA export factors renders cells highly permissive to influenza virus replication, demonstrating that proper levels of key constituents of the mRNA export machinery protect against influenza virus replication. Because Nup98 and Rae1 are induced by interferons, down-regulation of this pathway is likely a viral strategy to promote viral replication. These findings demonstrate previously undescribed influenza-mediated viral-host interactions and provide insights into potential molecular therapies that may interfere with influenza infection.

The evolutionarily conserved TRanscript-EXport (TREX) complex plays central roles during mRNP (messenger ribonucleoprotein) maturation and export from the nucleus to the cytoplasm. In yeast, TREX is composed of the THO sub-complex (Tho2, Hpr1, Tex1, Mft1, and Thp2), the DEAD box ATPase Sub2, and Yra1. Here we present a 3.7 Å cryo-EM structure of the yeast THO•Sub2 complex. The structure reveals the intimate assembly of THO revolving around its largest subunit Tho2. THO stabilizes a semi-open conformation of the Sub2 ATPase via interactions with Tho2. We show that THO interacts with the serine–arginine (SR)-like protein Gbp2 through both the RS domain and RRM domains of Gbp2. Cross-linking mass spectrometry analysis supports the extensive interactions between THO and Gbp2, further revealing that RRM domains of Gbp2 are in close proximity to the C-terminal domain of Tho2. We propose that THO serves as a landing pad to configure Gbp2 to facilitate its loading onto mRNP.

Hepatocellular carcinoma (HCC) is one of the most prevalent and deadly tumors; however, its pathogenic mechanism remains largely elusive. In-depth researches are needed to reveal the expression regulatory mechanisms and functions of the RNA-binding protein RALY in HCC. Here, we identify RALY as a highly expressed oncogenic factor that affects HCC cells proliferation both and . O-GlcNAcylation of RALY at Ser176 enhances its stability by protecting RALY from TRIM27-mediated ubiquitination, thus maintaining hyper-expression of the RALY protein. Mechanistically, RALY interacts with USP22 messenger RNA, as revealed by RNA immunoprecipitation, to increase their cytoplasmic localization and protein expression, thereby promoting the proliferation of HCC cells. Furthermore, we develop a novel RALY protein degrader based on peptide proteolysis-targeting chimeras, named RALY-PROTAC, which we chemically synthesize by linking a RALY-targeting peptide with the E3 ubiquitin ligase recruitment ligand pomalidomide. In conclusion, our findings demonstrate a novel mechanism by which O-GlcNAcylation/RALY/USP22 mRNA axis aggravates HCC cells proliferation. RALY-PROTACs as degraders of the RALY protein exhibit potential as therapeutic drugs for RALY-overexpressing HCC.

The circadian clock of plants allows them to cope with daily changes in their environment. This is accomplished by the rhythmic regulation of gene expression, in a process that involves many regulatory steps. One of the key steps involved at the RNA level is post-transcriptional regulation, which ensures a correct control on the different amounts and types of mRNA that will ultimately define the current physiological state of the plant cell. Recent advances in the study of the processes of regulation of pre-mRNA processing, RNA turn-over and surveillance, regulation of translation, function of lncRNAs, biogenesis and function of small RNAs, and the development of bioinformatics tools have helped to vastly expand our understanding of how this regulatory step performs its role. In this work we review the current progress in circadian regulation at the post-transcriptional level research in plants. It is the continuous interaction of all the information flow control post-transcriptional processes that allow a plant to precisely time and predict daily environmental changes.

Viruses are known for their ability to alter host gene expression. Kaposi sarcoma-associated herpesvirus has two proteins that obstruct host gene expression. KSHV SOX, encoded by the open reading frame 37 (ORF37), induces a widespread cytoplasmic mRNA degradation and a block on mRNA nuclear export. The other KSHV protein, encoded by the open reading frame 10 (ORF10), was recently identified to inhibit host gene expression through its direct function on the cellular mRNA export pathway. In this review, we summarize the studies on both SOX and ORF10 in efforts to elucidate their mechanisms. We also discuss how the findings based on a closely related rodent virus, murine gammaherpesvirus-68 (MHV-68), complement the KSHV findings to decipher the role of these two proteins in viral pathogenesis.

The eukaryotic translation initiation factor 4E (eIF4E) controls gene expression through its effects on mRNA export and cap‐dependent translation, both of which contribute to its oncogenic potential. In contrast to its translation function, the mRNA export function of eIF4E is poorly understood. Using an RNP isolation/mass spectrometry approach, we identified candidate cofactors of eIF4E mRNA export including LRPPRC. This protein associates with mRNAs containing the eIF4E‐sensitivity element (4E‐SE), and its overexpression alters the nuclear export of several eIF4E‐sensitive mRNAs. LRPPRC‐mediated alteration of eIF4E's mRNA export function requires the integrity of its eIF4E‐binding site and it coincides with the subcellular re‐distribution of eIF4E. The eIF4E export RNP is distinct in composition from the bulk mRNA export pathway, in that eIF4E‐ and eIF4E‐sensitive mRNAs do not associate with general mRNA export factors such as TAP/NXF1 or REF/Aly. Our data indicate that mRNA export pathways have evolved for specific mRNAs enabling the differential regulation of biochemical pathways by modulating the expression of groups of genes at the level of their export.

Influenza A viruses (IAV) utilize host proteins throughout their life cycle to infect and replicate in their hosts. We previously showed that host adaptive mutations in avian IAV PA help recruit host protein G-Rich RNA Sequence Binding Factor 1 (GRSF1) to the nucleoprotein (NP) 5’ untranslated region (UTR), leading to the enhanced nuclear export and translation of NP mRNA. In this study, we evaluated the impact of GRSF1 in the viral life cycle. We rescued and characterized a 2009 pH1N1 virus with a mutated GRSF1 binding site in the 5’ UTR of NP mRNA. Mutant viral growth was attenuated relative to pH1N1 wild-type (WT) in mammalian cells. We observed a specific reduction in the NP protein production and cytosolic accumulation of NP mRNAs, indicating a critical role of GRSF1 in the nuclear export of IAV NP mRNAs. Further, in vitro-transcribed mutated NP mRNA was translated less efficiently than WT NP mRNA in transfected cells. Together, these findings show that GRSF1 binding is important for both mRNA nuclear export and translation and affects overall IAV growth. Enhanced association of GRSF1 to NP mRNA by PA mutations leads to rapid virus growth, which could be a key process of mammalian host adaptation of IAV.

Nucleocytoplasmic transport (NCT) across thenuclear envelope (NE) is tightly regulated in eukaryotic cells and iscritical for maintaining cellular homeostasis. Its dysregulationleads to aging and neurodegeneration. Because they maintainaging-associated hallmarks, directly reprogrammed neurons from human fibroblasts are invaluable in understanding NCT. However, it is not clear whether NCT activity is influenced by neuronal maturation and sample sex [a key biological variable emphasized by the National Institutes of Health (NIH) policy]. We examined here NCT activity at the single-cell level by measuring mRNA subcellular distribution and protein transport in directly induced motor neurons (diMNs) from adult human fibroblasts. The results show that mRNA subcellular distribution but not protein transport is affected by neuronal maturation stages, whereas both transport processes are not influenced by the sample sex. This study also provides quantitative methods and optimized conditions for measuring NCTs of mRNAs or protein cargoes, establishing a robust way for future functional examinations of NCT activity in directly induced neurons from diseased human patients.

We asked whether the C-terminal repeat domain (CTD) kinase, CDK12/CyclinK, phosphorylates substrates in addition to the CTD of RPB1, using our CDK12analog-sensitive HeLa cell line to investigate CDK12 activity-dependent phosphorylation events in human cells. Characterizing the phospho-proteome before and after selective inhibition of CDK12 activity by the analog 1-NM-PP1, we identified 5,644 distinct phospho-peptides, among which were 50 whose average relative amount decreased more than 2-fold after 30 min of inhibition (none of these derived from RPB1). Half of the phospho-peptides actually showed >3-fold decreases, and a dozen showed decreases of 5-fold or more. As might be expected, the 40 proteins that gave rise to the 50 affected phospho-peptides mostly function in processes that have been linked to CDK12, such as transcription and RNA processing. However, the results also suggest roles for CDK12 in other events, notably mRNA nuclear export, cell differentiation and mitosis. While a number of the more-affected sites resemble the CTD in amino acid sequence and are likely direct CDK12 substrates, other highly-affected sites are not CTD-like, and their decreased phosphorylation may be a secondary (downstream) effect of CDK12 inhibition.