scholarly journals Consensus PP1 Binding Motifs Regulate Transcriptional Corepression and Alternative RNA Splicing Activities of the Steroid Receptor Coregulators, p54nrb and PSF

2011 ◽  
Vol 25 (7) ◽  
pp. 1197-1210 ◽  
Author(s):  
Liangliang Liu ◽  
Ning Xie ◽  
Paul Rennie ◽  
John R. G. Challis ◽  
Martin Gleave ◽  
...  
Keyword(s):  
Gene Transcription ◽  
Protein Interactions ◽  
Rna Splicing ◽  
Exon Skipping ◽  
Steroid Receptor ◽  
Splicing Factors ◽  
Histone Deacetylase 1 ◽  
Transcriptional Corepressors ◽  
Rvxf Motif

Abstract Originally identified as essential pre-mRNA splicing factors, non-POU-domain-containing, octamer binding protein (p54nrb) and PTB-associated RNA splicing factor (PSF) are also steroid receptor corepressors. The mechanisms by which p54nrb and PSF regulate gene transcription remain unclear. Both p54nrb and PSF contain protein phosphatase 1 (PP1) consensus binding RVxF motifs, suggesting that PP1 may regulate phosphorylation status of p54nrb and PSF and thus their function in gene transcription. In this report, we demonstrated that PP1 forms a protein complex with both p54nrb and PSF. PP1 interacts directly with the RVxF motif only in p54nrb, but not in PSF. Association with PP1 results in dephosphorylation of both p54nrb and PSF in vivo and the loss of their transcriptional corepressor activities. Using the CD44 minigene as a reporter, we showed that PP1 regulates p54nrb and PSF alternative splicing activities that determine exon skipping vs. inclusion in the final mature RNA for translation. In addition, changes in transcriptional corepression and RNA splicing activities of p54nrb and PSF are correlated with alterations in protein interactions of p54nrb and PSF with transcriptional corepressors such as Sin3A and histone deacetylase 1, and RNA splicing factors such as U1A and U2AF. Furthermore, we demonstrated a novel function of the RVxF motif within PSF that enhances its corepression and RNA splicing activities independent of PP1. We conclude that the RVxF motifs play an important role in controlling the multifunctional properties of p54nrb and PSF in the regulation of gene transcription.

Splicing signals and factors in plant intron removal

2002 ◽  
Vol 30 (2) ◽  
pp. 146-149 ◽  
Author(s):  
J. W. S. Brown ◽  
C. G. Simpson ◽  
G. Thow ◽  
G. P. Clark ◽  
S. N. Jennings ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Viral Vectors ◽  
Binding Proteins ◽  
Fluorescent Protein ◽  
Splicing Factors ◽  
Small Nuclear Rna ◽  
Intron Removal ◽  
Arabidopsis Proteins ◽  
Green Fluorescent

Constitutive splicing of the potato invertase miniexon 2 (9 nt long) requires a branchpoint sequence positioned around 50 nt upstream of the 5′ splice site of the adjacent intron and a U11 element found just downstream of the branchpoint in the upstream intron [Simpson, Hedley, Watters, Clark, McQuade, Machray and Brown (2000) RNA 6, 422–433]. The sensitivity of this in vivo plant splicing system has been used to demonstrate exon scanning in plants, and to characterize plant intronic elements, such as branchpoint and poly-pyrimidine tract sequences. Plant introns differ from their vertebrate and yeast couterparts in being UA- or U-rich (up to 85% UA). One of the key differences in splicing between plants and other eukaryotes lies in early intron recognition, which is thought to be mediated by UA-binding proteins. We are adopting three approaches to studying the RNA-protein interactions in plant splicing. First, overexpression of plant splicing factors and, in particular, UA-binding proteins, in conjunction with a range of mini-exon mutants. Secondly, the sequences of around 65% of vertebrate and yeast splicing factors have high-quality matches to Arabidopsis proteins, opening the door to identification and analysis of gene knockouts. Finally, to discover plant-specific proteins involved in splicing and in, for example, rRNA or small nuclear RNA processing, green fluorescent protein-cDNA fusion libraries in viral vectors are being screened.

scholarly journals In vivo recognition of a vertebrate mini-exon as an exon-intron-exon unit.

1993 ◽  
Vol 13 (5) ◽  
pp. 2677-2687 ◽  
Author(s):  
D A Sterner ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Troponin I ◽  
Exon Skipping ◽  
Splice Sites ◽  
Small Vertebrate ◽  
Upstream Exon ◽  
I Gene

Very small vertebrate exons are problematic for RNA splicing because of the proximity of their 3' and 5' splice sites. In this study, we investigated the recognition of a constitutive 7-nucleotide mini-exon from the troponin I gene that resides quite close to the adjacent upstream exon. The mini-exon failed to be included in spliced RNA when placed in a heterologous gene unless accompanied by the upstream exon. The requirement for the upstream exon disappeared when the mini-exon was internally expanded, suggesting that the splice sites bordering the mini-exon are compatible with those of other constitutive vertebrate exons and that the small size of the exon impaired inclusion. Mutation of the 5' splice site of the natural upstream exon did not result in either exon skipping or activation of a cryptic 5' splice site, the normal vertebrate phenotypes for such mutants. Instead, a spliced RNA accumulated that still contained the upstream intron. In vitro, the mini-exon failed to assemble into spliceosome complexes unless either internally expanded or accompanied by the upstream exon. Thus, impaired usage of the mini-exon in vivo was accompanied by impaired recognition in vitro, and recognition of the mini-exon was facilitated by the presence of the upstream exon in vivo and in vitro. Cumulatively, the atypical in vivo and in vitro properties of the troponin exons suggest a mechanism for the recognition of this mini-exon in which initial recognition of an exon-intron-exon unit is followed by subsequent recognition of the intron.

scholarly journals Efficacy of longevity interventions in C. elegans is determined by early life activity of RNA splicing factors

2021 ◽  
Author(s):  
Sneha Dutta ◽  
Caroline Heintz ◽  
Maria C. Perez-Matos ◽  
Ayse Sena Mutlu ◽  
Mary E Piper ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Early Life ◽  
Late Onset ◽  
Natural Aging ◽  
Splicing Factors ◽  
Life Activity ◽  
C Elegans ◽  
Lipid Utilization ◽  
Early Inhibition

Geroscience aims to target the aging process to extend healthspan. However, even isogenic individuals show heterogeneity in natural aging rate and responsiveness to pro-longevity interventions, limiting translational potential. Using in vivo mini gene reporters in isogenic C. elegans, we show that alternative splicing of mRNAs related to lipid metabolism in young animals is coupled to subsequent life expectancy. Further, activity of RNA splicing factors REPO-1 and SFA-1 early in life modulates effectiveness of specific longevity interventions via POD-2/ACC1 and regulation of lipid utilization. In addition, early inhibition of REPO-1 renders animals refractory to late onset suppression of the TORC1 pathway. Together these data suggest that activity of RNA splicing factors and the metabolic landscape early in life can modulate responsiveness to longevity interventions and may explain variance in efficacy between individuals.

In vivo recognition of a vertebrate mini-exon as an exon-intron-exon unit

1993 ◽  
Vol 13 (5) ◽  
pp. 2677-2687
Author(s):  
D A Sterner ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Troponin I ◽  
Exon Skipping ◽  
Splice Sites ◽  
Small Vertebrate ◽  
Upstream Exon ◽  
I Gene

Very small vertebrate exons are problematic for RNA splicing because of the proximity of their 3' and 5' splice sites. In this study, we investigated the recognition of a constitutive 7-nucleotide mini-exon from the troponin I gene that resides quite close to the adjacent upstream exon. The mini-exon failed to be included in spliced RNA when placed in a heterologous gene unless accompanied by the upstream exon. The requirement for the upstream exon disappeared when the mini-exon was internally expanded, suggesting that the splice sites bordering the mini-exon are compatible with those of other constitutive vertebrate exons and that the small size of the exon impaired inclusion. Mutation of the 5' splice site of the natural upstream exon did not result in either exon skipping or activation of a cryptic 5' splice site, the normal vertebrate phenotypes for such mutants. Instead, a spliced RNA accumulated that still contained the upstream intron. In vitro, the mini-exon failed to assemble into spliceosome complexes unless either internally expanded or accompanied by the upstream exon. Thus, impaired usage of the mini-exon in vivo was accompanied by impaired recognition in vitro, and recognition of the mini-exon was facilitated by the presence of the upstream exon in vivo and in vitro. Cumulatively, the atypical in vivo and in vitro properties of the troponin exons suggest a mechanism for the recognition of this mini-exon in which initial recognition of an exon-intron-exon unit is followed by subsequent recognition of the intron.

scholarly journals Tax Interacts with P-TEFb in a Novel Manner To Stimulate Human T-Lymphotropic Virus Type 1 Transcription

2006 ◽  
Vol 80 (10) ◽  
pp. 4781-4791 ◽  
Author(s):  
Meisheng Zhou ◽  
Hanxin Lu ◽  
Hyeon Park ◽  
Jaime Wilson-Chiru ◽  
Rebecca Linton ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Virus Type ◽  
Protein Interactions ◽  
Elongation Factor ◽  
Protein Protein Interactions ◽  
T Lymphotropic Virus ◽  
Histone Deacetylase 1

ABSTRACT Human T-lymphotropic virus type 1 (HTLV-1) encodes a transcriptional activator, Tax, whose function is essential for viral transcription and replication. Tax transactivates the viral long-terminal repeat through a series of protein-protein interactions which facilitate CREB and CBP/p300 binding. In addition, Tax dissociates transcription repressor histone deacetylase 1 interaction with the CREB response element. The subsequent events through which Tax interacts and communicates with RNA polymerase II and cyclin-dependent kinases (CDKs) are not clearly understood. Here we present evidence that Tax recruits positive transcription elongation factor b (P-TEFb) (CDK9/cyclin T1) to the viral promoter. This recruitment likely involves protein-protein interactions since Tax associates with P-TEFb in vitro as demonstrated by glutathione S-transferase fusion protein pull-down assays and in vivo as shown by coimmunoprecipitation assays. Functionally, small interfering RNA directed toward CDK9 inhibited Tax transactivation in transient assays. Consistent with these findings, the depletion of CDK9 from nuclear extracts inhibited Tax transactivation in vitro. Reconstitution of the reaction with wild-type P-TEFb, but not a kinase-dead mutant, recovered HTLV-1 transcription. Moreover, the addition of the CDK9 inhibitor flavopiridol blocked Tax transactivation in vitro and in vivo. Interestingly, we found that Tax regulates CDK9 kinase activity through a novel autophosphorylation pathway.

scholarly journals SFMetaDB: A Comprehensive Annotation of Mouse RNA Splicing Factor RNA-Seq Datasets

2017 ◽  
Author(s):  
Jin Li ◽  
Ching-San Tseng ◽  
Antonio Federico ◽  
Franjo Ivankovic ◽  
Yi-Shuian Huang ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Pfam Domain ◽  
Research Direction ◽  
Splicing Factors ◽  
Rna Seq ◽  
Functional Studies ◽  
Fruitful Research ◽  
Potential Use

AbstractAlthough the number of RNA-Seq datasets deposited publicly has increased over the past few years, incomplete annotation of the associated metadata limits their potential use. Because of the importance of RNA splicing in diseases and biological processes, we constructed a database called SFMetaDB by curating datasets related with RNA splicing factors. Our effort focused on the RNA-Seq datasets in which splicing factors were knocked-down, knocked-out or over-expressed, leading to 75 datasets corresponding to 56 splicing factors. These datasets can be used in differential alternative splicing analysis for the identification of the potential targets of these splicing factors and other functional studies. Surprisingly, only ∼15% of all the splicing factors have been studied by loss- or gain-of-function experiments using RNA-Seq. In particular, splicing factors with domains from a few dominant Pfam domain families have not been studied. This suggests a significant gap that needs to be addressed to fully elucidate the splicing regulatory landscape. Indeed, there are already mouse models available for ∼20 of the unstudied splicing factors, and it can be a fruitful research direction to study these splicing factors in vitro and in vivo using RNA-Seq.Database URLhttp://sfmetadb.ece.tamu.edu/

scholarly journals SON drives oncogenic RNA splicing in glioblastoma by regulating PTBP1/PTBP2 switching and RBFOX2 activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jung-Hyun Kim ◽  
Kyuho Jeong ◽  
Jianfeng Li ◽  
James M. Murphy ◽  
Lana Vukadin ◽  
...  
Keyword(s):  
Glioblastoma Multiforme ◽  
Cancer Therapy ◽  
Brain Tumors ◽  
Rna Splicing ◽  
Splicing Factors ◽  
Potential Therapeutic Target ◽  
Intron Removal ◽  
Orthotopic Xenografts

AbstractWhile dysregulation of RNA splicing has been recognized as an emerging target for cancer therapy, the functional significance of RNA splicing and individual splicing factors in brain tumors is poorly understood. Here, we identify SON as a master regulator that activates PTBP1-mediated oncogenic splicing while suppressing RBFOX2-mediated non-oncogenic neuronal splicing in glioblastoma multiforme (GBM). SON is overexpressed in GBM patients and SON knockdown causes failure in intron removal from the PTBP1 transcript, resulting in PTBP1 downregulation and inhibition of its downstream oncogenic splicing. Furthermore, SON forms a complex with hnRNP A2B1 and antagonizes RBFOX2, which leads to skipping of RBFOX2-targeted cassette exons, including the PTBP2 neuronal exon. SON knockdown inhibits proliferation and clonogenicity of GBM cells in vitro and significantly suppresses tumor growth in orthotopic xenografts in vivo. Collectively, our study reveals that SON-mediated RNA splicing is a GBM vulnerability, implicating SON as a potential therapeutic target in brain tumors.

scholarly journals Retinoblastoma and the Related Pocket Protein p107 Act as Coactivators of NeuroD1 to Enhance Gene Transcription

2005 ◽  
Vol 280 (16) ◽  
pp. 16088-16095 ◽  
Author(s):  
Eric Batsché ◽  
Pandelis Moschopoulos ◽  
Julien Desroches ◽  
Steve Bilodeau ◽  
Jacques Drouin
Keyword(s):  
Gene Transcription ◽  
Protein Interactions ◽  
Mrna Levels ◽  
Orphan Nuclear Receptor ◽  
Knock Out ◽  
Helix Loop Helix ◽  
Knock Out Mice ◽  
Interfering Rna

Gene inactivation studies have suggested that the product of the retinoblastoma gene,Rb, is particularly limiting in pituitary pro-opiomelanocortin (POMC)-expressing cell lineages. Indeed, in Rb knock-out mice, these cells develop tumors with high frequency. To understand the implication of limiting Rb expression in these cells, we investigated the action of Rb and its related pocket proteins, p107 and p130, onPOMCgene transcription. This led to the identification of the neurogenic basic helix-loop-helix transcription factor, NeuroD1, as a target of Rb action. Rb and to a lesser extent p107, but not p130, enhance NeuroD1-dependent transcription, and this activity appears to depend on direct protein interactions between the Rb pocket and the helix-loop-helix domain of NeuroD1.In vivo, NeuroD is found in a complex that includes Rb and also the orphan nuclear receptor NGFI-B, which mediates corticotropin-releasing hormone activation of POMC transcription. The formation of a similar complexin vitrorequires the presence of Rb as a bridge between NeuroD and NGFI-B. In POMC-expressing AtT-20 cells, Rb and p107 are present on the POMC promoter and inhibition of their expression through small interfering RNA decreases POMC mRNA levels. The action of Rb and its related proteins on POMC transcription may contribute to the establishment and/or maintenance of the differentiation phenotype.

scholarly journals Secondary motifs enable concentration-dependent regulation by Rbfox family proteins

2019 ◽  
Author(s):  
Bridget E. Begg ◽  
Marvin Jens ◽  
Peter Y. Wang ◽  
Christopher B. Burge
Keyword(s):  
Alternative Splicing ◽  
Rna Splicing ◽  
Neuronal Development ◽  
Splicing Factors ◽  
Reporter Assay ◽  
Animal Development ◽  
High Affinity ◽  
Second Wave ◽  
Relevant Range

AbstractThe Rbfox family of splicing factors regulate alternative splicing during animal development and in disease, impacting thousands of exons in the maturing brain, heart, and muscle. Rbfox proteins have long been known to bind to the RNA sequence GCAUG with high affinity, but just half of Rbfox CLIP peaks contain a GCAUG motif. We incubated recombinant RBFOX2 with over 60,000 transcriptomic sequences to reveal significant binding to several moderate-affinity, non-GCAYG sites at a physiologically relevant range of RBFOX concentrations. We find that many of these “secondary motifs” bind Rbfox robustly in vivo and that several together can exert regulation comparable to a GCAUG in a trichromatic splicing reporter assay. Furthermore, secondary motifs regulate RNA splicing in neuronal development and in neuronal subtypes where cellular Rbfox concentrations are highest, enabling a second wave of splicing changes as Rbfox levels increase.

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