scholarly journals The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Saikat Bhattacharya ◽  
Michaella J. Levy ◽  
Ning Zhang ◽  
Hua Li ◽  
Laurence Florens ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Rna Binding ◽  
Mrna Processing ◽  
Key Factors ◽  
Pol Ii ◽  
Mrna Transcripts ◽  
Hnrnp Protein ◽  
Processing Factors

AbstractHeterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2, by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.

scholarly journals The histone methyltransferase SETD2 couples transcription and splicing by engaging pre-mRNA processing factors through its SHI domain

2020 ◽  
Author(s):  
Saikat Bhattacharya ◽  
Michaella J. Levy ◽  
Ning Zhang ◽  
Hua Li ◽  
Laurence Florens ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Rna Binding ◽  
Histone Methyltransferase ◽  
Mrna Processing ◽  
Key Factors ◽  
Mrna Transcripts ◽  
Hnrnp Protein ◽  
Processing Factors

SUMMARYHeterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP L Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2 by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.

scholarly journals Coupling of Termination, 3′ Processing, and mRNA Export

2002 ◽  
Vol 22 (18) ◽  
pp. 6441-6457 ◽  
Author(s):  
C. M. Hammell ◽  
Stefan Gross ◽  
Daniel Zenklusen ◽  
Catherine V. Heath ◽  
Francoise Stutz ◽  
...  
Keyword(s):  
Mrna Export ◽  
Mrna Processing ◽  
Nonpermissive Temperature ◽  
Pol Ii ◽  
The Core ◽  
In Vitro Processing ◽  
Novel Alleles ◽  
Processing Factors

ABSTRACT In a screen to identify genes required for mRNA export in Saccharomyces cerevisiae, we isolated an allele of poly(A) polymerase (PAP1) and novel alleles encoding several other 3′ processing factors. Many newly isolated and some previously described mutants (rna14-48, rna14-49, rna14-64, rna15-58, and pcf11-1 strains) are defective in polymerase II (Pol II) termination but, interestingly, retain the ability to polyadenylate these improperly processed transcripts at the nonpermissive temperature. Deletion of the cis-acting sequences required to couple 3′ processing and termination also produces transcripts that fail to exit the nucleus, suggesting that all of these processes (cleavage, termination, and export) are coupled. We also find that several but not all mRNA export mutants produce improperly 3′ processed transcripts at the nonpermissive temperature. 3′ maturation defects in mRNA export mutants include improper Pol II termination and/or the previously characterized hyperpolyadenylation of transcripts. Importantly, not all mRNA export mutants have defects in 3′ processing. The similarity of the phenotypes of some mRNA export mutants and 3′ processing mutants indicates that some factors from each process may mechanistically interact to couple mRNA processing and export. Consistent with this assumption, we present evidence that Xpo1p interacts in vivo with several 3′ processing factors and that the addition of recombinant Xpo1p to in vitro processing reaction mixtures stimulates 3′ maturation. Of the core 3′ processing factors tested (Rna14p, Rna15p, Pcf11p, Hrp1p, Fip1p, and Cft1p), only Hrp1p shuttles. Overexpression of Rat8p/Dbp5p suppresses both 3′ processing and mRNA export defects found in xpo1-1 cells.

scholarly journals High-resolution NMR structures of the domains ofSaccharomyces cerevisiaeTho1

2016 ◽  
Vol 72 (6) ◽  
pp. 500-506
Author(s):  
Julian O. B. Jacobsen ◽  
Mark D. Allen ◽  
Stefan M. V. Freund ◽  
Mark Bycroft
Keyword(s):  
High Resolution ◽  
Rna Binding ◽  
Mrna Processing ◽  
Dependent Manner ◽  
Nmr Structures ◽  
High Resolution Nmr ◽  
N Terminus ◽  
Resolution Structure

THO is a multi-protein complex involved in the formation of messenger ribonuclear particles (mRNPs) by coupling transcription with mRNA processing and export. THO is thought to be formed from five subunits, Tho2p, Hpr1p, Tex1p, Mft1p and Thp2p, and recent work has determined a low-resolution structure of the complex [Poulsenet al.(2014),PLoS One,9, e103470]. A number of additional proteins are thought to be involved in the formation of mRNP in yeast, including Tho1, which has been shown to bind RNAin vitroand is recruited to actively transcribed chromatinin vivoin a THO-complex and RNA-dependent manner. Tho1 is known to contain a SAP domain at the N-terminus, but the ability to suppress the expression defects of thehpr1Δ mutant of THO was shown to reside in the RNA-binding C-terminal region. In this study, high-resolution structures of both the N-terminal DNA-binding SAP domain and C-terminal RNA-binding domain have been determined.

scholarly journals Drosophila Stem-Loop Binding Protein Intracellular Localization Is Mediated by Phosphorylation and Is Required for Cell Cycle-regulated Histone mRNA Expression

2004 ◽  
Vol 15 (3) ◽  
pp. 1112-1123 ◽  
Author(s):  
David J. Lanzotti ◽  
Jeremy M. Kupsco ◽  
Xiao-Cui Yang ◽  
Zbigniew Dominski ◽  
William F. Marzluff ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Binding ◽  
Binding Protein ◽  
Intracellular Localization ◽  
Mrna Processing ◽  
Nuclear Targeting ◽  
Histone Mrna ◽  
Stem Loop

Stem-loop binding protein (SLBP) is an essential component of the histone pre-mRNA processing machinery. SLBP protein expression was examined during Drosophila development by using transgenes expressing hemagglutinin (HA) epitope-tagged proteins expressed from the endogenous Slbp promoter. Full-length HA-dSLBP complemented a Slbp null mutation, demonstrating that it was fully functional. dSLBP protein accumulates throughout the cell cycle, in contrast to the observed restriction of mammalian SLBP to S phase. dSLBP is located in both nucleus and cytoplasm in replicating cells, but it becomes predominantly nuclear during G2. dSLBP is present in mitotic cells and is down-regulated in G1 when cells exit the cell cycle. We determined whether mutation at previously identified phosphorylation sites, T120 and T230, affected the ability of the protein to restore viability and histone mRNA processing to dSLBP null mutants. The T120A SLBP restored viability and histone pre-mRNA processing. However, the T230A mutant, located in a conserved TPNK sequence in the RNA binding domain, did not restore viability and histone mRNA processing in vivo, although it had full activity in histone mRNA processing in vitro. The T230A protein is concentrated in the cytoplasm, suggesting that it is defective in nuclear targeting, and accounting for its failure to function in histone pre-mRNA processing in vivo.

Abstract 355: In Vivo Targeting Of GTF2B Selectively Inhibits Transcription Of Cardiomyopathy-related Genes And Partially Blunts Development Of Cardiac Hypertrophy

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Danish Sayed ◽  
Zhi Yang ◽  
Minzhen He ◽  
Maha Abdellatif
Keyword(s):  
Rna Polymerase Ii ◽  
Rna Splicing ◽  
De Novo ◽  
Transcriptional Profiling ◽  
Essential Genes ◽  
Pol Ii ◽  
Expression Of Genes ◽  
Essential Components

Transcriptional profiling of cardiac genome during hypertrophy identified two categories of genes with distinct modes of regulation. The first set of genes involved in the cells essential functions (e.g. RNA splicing) and whose transcription is expected to be incremental and contribute to the increasing cardiac mass is regulated by promoter clearance of RNA polymerase II (pol II). On the other hand, the second set that include genes with specialized function and show a robust increase in expression upon growth stimulus (cytoskeletal, extracellular matrix) are regulated by de novo pol II recruitment to promoters. Our goal was to identify the transcriptional mechanisms that distinguish these two sets of genes and then to selectively inhibit those that participate in contractile dysfunction, while preserving the expression of genes necessary for essential functions. General Transcription factor IIB (GTF2B), is one of the essential components of transcription machinery and is required for pol II recruitment. Thus, we hypothesized that inhibition of GTF2B would result in inhibition of only the specialized genes, sparing the essential genes. Our in vitro results with shRNA mediated inhibition of GTF2B in hypertrophying neonatal myocytes showed decreased expression of genes that required de novo pol II recruitment for transcription (eg. ACTA1), while no change was observed in the genes regulated by promoter clearance of pol II (Vdac1). Similarly, preliminary results with in vivo knockdown of GTF2B (~80% reduction in mRNA and ~36% in protein) via intravenous injection of modified antisense oligo in mice subjected to transaortic coarctation (TAC) showed inhibition of only cardiomyopathy-related genes that require pol II recruitment (ANF), while expression of essential genes (Vdac1) remained unchanged. Inhibition of GTF2B restricted increase in TAC-induced heart wt to 9%, compared to 29% in TAC hearts with control oligo. Echocardiography showed partial normalization of ejection fraction with GTF2B inhibitor during TAC from 61.5% to 66.4% compared to sham hearts with 71%. Thus, we conclude that by targeting GTF2B we can selectively restrict the expression of detrimental genes during hypertrophy, thereby delaying the onset of cardiac dysfunction and failure

scholarly journals SRm160 Splicing Coactivator Promotes Transcript 3′-End Cleavage

2002 ◽  
Vol 22 (1) ◽  
pp. 148-160 ◽  
Author(s):  
Susan McCracken ◽  
Mark Lambermon ◽  
Benjamin J. Blencowe
Keyword(s):  
Gene Expression ◽  
Matrix Protein ◽  
Mrna Processing ◽  
Nuclear Matrix Protein ◽  
Mrna Transcripts ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Cytoplasmic Accumulation

ABSTRACT Individual steps in the processing of pre-mRNA, including 5′-end cap formation, splicing, and 3′-end processing (cleavage and polyadenylation) are highly integrated and can influence one another. In addition, prior splicing can influence downstream steps in gene expression, including export of mRNA from the nucleus. However, the factors and mechanisms coordinating these steps in the maturation of pre-mRNA transcripts are not well understood. In the present study we demonstrate that SRm160 (for serine/arginine repeat-related nuclear matrix protein of 160 kDa), a coactivator of constitutive and exon enhancer-dependent splicing, participates in 3′-end formation. Increased levels of SRm160 promoted the 3′-end cleavage of transcripts both in vivo and in vitro. Remarkably, at high levels in vivo SRm160 activated the 3′-end cleavage and cytoplasmic accumulation of unspliced pre-mRNAs, thereby uncoupling the requirement for splicing to promote the 3′-end formation and nuclear release of these transcripts. Consistent with a role in 3′-end formation coupled to splicing, SRm160 was found to associate specifically with the cleavage polyadenylation specificity factor and to stimulate the 3′-end cleavage of splicing-active pre-mRNAs more efficiently than that of splicing-inactive pre-mRNAs in vitro. The results provide evidence for a role for SRm160 in mRNA 3′-end formation and suggest that the level of this splicing coactivator is important for the proper coordination of pre-mRNA processing events.

scholarly journals GPKOW is essential for pre-mRNA splicing in vitro and suppresses splicing defect caused by dominant-negative DHX16 mutation in vivo

2014 ◽  
Vol 34 (6) ◽  
Author(s):  
Shengbing Zang ◽  
Ting-Yu Lin ◽  
Xinji Chen ◽  
Marieta Gencheva ◽  
Alain N. S. Newo ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Mrna Splicing ◽  
Dominant Negative ◽  
Splicing Defect ◽  
Similarity And Difference

Using biochemical, mutation, and cellular analyses, we characterized important domains involved in the functionality of a RNA-binding protein in RNA splicing. We also showed the similarity and difference between yeast and human counterparts.

scholarly journals Role for PSF in Mediating Transcriptional Activator-Dependent Stimulation of Pre-mRNA Processing In Vivo

2005 ◽  
Vol 25 (15) ◽  
pp. 6734-6746 ◽  
Author(s):  
Emanuel Rosonina ◽  
Joanna Y. Y. Ip ◽  
John A. Calarco ◽  
Malina A. Bakowski ◽  
Andrew Emili ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Mrna Processing ◽  
Pol Ii ◽  
293 Cells ◽  
Two Factors ◽  
Elevated Expression ◽  
Strong Activator ◽  
Stimulation Of

ABSTRACT In a recent study, we provided evidence that strong promoter-bound transcriptional activators result in higher levels of splicing and 3′-end cleavage of nascent pre-mRNA than do weak promoter-bound activators and that this effect of strong activators requires the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II). In the present study, we have investigated the mechanism of activator- and CTD-mediated stimulation of pre-mRNA processing. Affinity chromatography experiments reveal that two factors previously implicated in the coupling of transcription and pre-mRNA processing, PSF and p54nrb/NonO, preferentially bind a strong rather than a weak activation domain. Elevated expression in human 293 cells of PSF bypasses the requirement for a strong activator to promote efficient splicing and 3′-end cleavage. Truncation of the pol II CTD, which consists of 52 repeats of the consensus heptapeptide sequence YSPTSPS, to 15 heptapeptide repeats prevents PSF-dependent stimulation of splicing and 3′-end cleavage. Moreover, PSF and p54nrb/NonO bind in vitro to the wild-type CTD but not to the truncated 15-repeat CTD, and domains in PSF that are required for binding to activators and to the CTD are also important for the stimulation of pre-mRNA processing. Interestingly, activator- and CTD-dependent stimulation of splicing mediated by PSF appears to primarily affect the removal of first introns. Collectively, these results suggest that the recruitment of PSF to activated promoters and the pol II CTD provides a mechanism by which transcription and pre-mRNA processing are coordinated within the cell.

scholarly journals Binding of a cell-type-specific RNA splicing factor to its target regulatory sequence.

1995 ◽  
Vol 15 (4) ◽  
pp. 1953-1960 ◽  
Author(s):  
K Nandabalan ◽  
G S Roeder
Keyword(s):  
Rna Splicing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Regulatory Element ◽  
Regulatory Sequence ◽  
Mobility Shift ◽  
Gel Mobility Shift ◽  
Hybrid Genes

The transcript of the Saccharomyces cerevisiae MER2 gene is spliced efficiently during meiosis but not during vegetative growth. Efficient splicing of the wild-type MER2 transcript requires the Mer1 protein, which is produced only in meiotic cells. Analysis of deletion and substitution mutations in the MER2 5' exon demonstrates that the unusually large size of this exon plays an important role in splicing regulation. The cis-acting sequences essential for Mer1-dependent splicing of MER2 RNA were determined by the analysis of MER2 deletion mutants and hybrid genes. The 80-base MER2 intron is sufficient for Mer1-dependent splicing in vivo, but sequences in the 5' exon enhance splicing efficiency. The Mer1 protein contains the KH motif found in some RNA-binding proteins, and RNA gel mobility shift assays demonstrate that Mer1 binds specifically to MER2 RNA. Both the transcript derived from the intronless MER2 gene and the transcript consisting only of the intron are able to bind to Mer1 in vitro, but neither has as high affinity for the protein as the intact substrate. RNase T1 footprinting indicates that the Mer1 protein contacts MER2 RNA at several points in the 5' exon and in the intron. Thus, Mer1 interacts directly with a regulatory element in MER2 RNA and promotes splicing.

Electromagnetic field in Alzheimer’s disease: a literature review of recent preclinical and clinical studies

2020 ◽  
Vol 17 ◽  
Author(s):  
Reem Habib Mohamad Ali Ahmad ◽  
Marc Fakhoury ◽  
Nada Lawand
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Neurodegenerative Disorder ◽  
In Vivo Studies ◽  
Key Factors ◽  
Potential Benefits ◽  
Preclinical And Clinical Studies ◽  
The Brain

: Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the progressive loss of neurons leading to cognitive and memory decay. The main signs of AD include the irregular extracellular accumulation of amyloidbeta (Aβ) protein in the brain and the hyper-phosphorylation of tau protein inside neurons. Changes in Aβ expression or aggregation are considered key factors in the pathophysiology of sporadic and early-onset AD and correlate with the cognitive decline seen in patients with AD. Despite decades of research, current approaches in the treatment of AD are only symptomatic in nature and are not effective in slowing or reversing the course of the disease. Encouragingly, recent evidence revealed that exposure to electromagnetic fields (EMF) can delay the development of AD and improve memory. This review paper discusses findings from in vitro and in vivo studies that investigate the link between EMF and AD at the cellular and behavioural level, and highlights the potential benefits of EMF as an innovative approach for the treatment of AD.

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