scholarly journals The Splicing Factor U2AF Small Subunit Is Functionally Conserved between Fission Yeast and Humans

2004 ◽  
Vol 24 (10) ◽  
pp. 4229-4240 ◽  
Author(s):  
Christopher J. Webb ◽  
Jo Ann Wise
Keyword(s):  
Rna Binding ◽  
Mutational Analysis ◽  
Large Subunit ◽  
Time Frame ◽  
Small Subunit ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Binding Domains ◽  
Site Recognition

ABSTRACT The small subunit of U2AF, which functions in 3′ splice site recognition, is more highly conserved than its heterodimeric partner yet is less thoroughly investigated. Remarkably, we find that the small subunit of Schizosaccharomyces pombe U2AF (U2AFSM) can be replaced in vivo by its human counterpart, demonstrating that the conservation extends to function. Precursor mRNAs accumulate in S. pombe following U2AFSM depletion in a time frame consistent with a role in splicing. A comprehensive mutational analysis reveals that all three conserved domains are required for viability. Notably, however, a tryptophan in the pseudo-RNA recognition motif implicated in a key contact with the large subunit by crystallographic data is dispensable whereas amino acids implicated in RNA recognition are critical. Mutagenesis of the two zinc-binding domains demonstrates that they are neither equivalent nor redundant. Finally, two- and three-hybrid analyses indicate that mutations with effects on large-subunit interactions are rare whereas virtually all alleles tested diminished RNA binding by the heterodimer. In addition to demonstrating extraordinary conservation of U2AF small-subunit function, these results provide new insights into the roles of individual domains and residues.

TLS (FUS) binds RNA in vivo and engages in nucleo-cytoplasmic shuttling

1997 ◽  
Vol 110 (15) ◽  
pp. 1741-1750 ◽  
Author(s):  
H. Zinszner ◽  
J. Sok ◽  
D. Immanuel ◽  
Y. Yin ◽  
D. Ron
Keyword(s):  
Rna Binding ◽  
Cellular Transformation ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Nucleocytoplasmic Shuttling ◽  
Uv Crosslinking ◽  
Recognition Motif ◽  
Specific Analysis ◽  
Human And Mouse

TLS, the product of a gene commonly translocated in liposarcomas (TLS), is prototypical of a newly identified class of nuclear proteins that contain a C-terminal domain with a distinct RNA recognition motif (RRM) surrounded by Arg-Gly-Gly (RGG) repeats. Its unique N terminus serves as an essential transforming domain for a number of fusion oncoproteins in human sarcomas and leukemias. In this study we use an in vivo UV crosslinking procedure to probe the interactions of TLS with RNA. TLS is found to bind RNA in vivo and the association of TLS with RNA is rapidly diminished by treating cells with transcriptional inhibitors. This suggests that the species bound by TLS turns over rapidly. Surprisingly, the RRM was found to be dispensable for RNA binding by TLS in vivo, suggesting that at any one time most of the interactions between TLS and RNA in the cell are not sequence specific. Analysis of inter specific heterokaryons formed between human and mouse or Xenopus cells revealed that TLS engages in rapid nucleocytoplasmic shuttling, a finding confirmed by the ability of anti-TLS antibodies to trap TLS when injected into the cytoplasm of HeLa cells. Cellular fractionation experiments suggest that TLS binds to RNA in both the nucleus and cytoplasm and support the hypothesis that TLS functions as a heterogeneous ribonuclear protein (hnRNP)-like chaperone of RNA. These findings are discussed in the context of the role altered forms of TLS play in cellular transformation.

Structure of the mRNA splicing complex component Cwc2: insights into RNA recognition

2011 ◽  
Vol 441 (2) ◽  
pp. 591-597 ◽  
Author(s):  
Peilong Lu ◽  
Guifeng Lu ◽  
Chuangye Yan ◽  
Li Wang ◽  
Wenqi Li ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Hydrophobic Interactions ◽  
Mrna Splicing ◽  
Regulatory Function ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Core Domain ◽  
Intron Removal

The Prp19-associated complex [NTC (nineteen complex)] plays a crucial role in intron removal during premature mRNA splicing in eukaryotes. Only one component of the NTC, Cwc2, is capable of binding RNA. In the present study we report the 1.9 Å (1 Å=0.1 nm) X-ray structure of the Cwc2 core domain, which is both necessary and sufficient for RNA binding. The Cwc2 core domain contains two sub-domains, a CCCH-type ZnF (zinc finger) and a RRM (RNA recognition motif). Unexpectedly, the ZnF domain and the RRM form a single folding unit, glued together by extensive hydrophobic interactions and hydrogen bonds. Structure-guided mutational analysis revealed that the intervening loop [known as the RB loop (RNA-binding loop)] between ZnF and RRM plays an essential role in RNA binding. In addition, a number of highly conserved positively charged residues on the β-strands of RRM make an important contribution to RNA binding. Intriguingly, these residues and a portion of the RB loop constitute an extended basic surface strip that encircles Cwc2 halfway. The present study serves as a framework for understanding the regulatory function of the NTC in RNA splicing.

scholarly journals RNA Binding by Histone Methyltransferases Set1 and Set2

2017 ◽  
Vol 37 (14) ◽  
Author(s):  
Camille Sayou ◽  
Gonzalo Millán-Zambrano ◽  
Helena Santos-Rosa ◽  
Elisabeth Petfalski ◽  
Samuel Robson ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
High Throughput Sequencing ◽  
Rna Binding ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Content Type ◽  
Histone Methyltransferases ◽  
Intergenic Spacers ◽  
High Enrichment

ABSTRACT Histone methylation at H3K4 and H3K36 is commonly associated with genes actively transcribed by RNA polymerase II (RNAPII) and is catalyzed by Saccharomyces cerevisiae Set1 and Set2, respectively. Here we report that both methyltransferases can be UV cross-linked to RNA in vivo. High-throughput sequencing of the bound RNAs revealed strong Set1 enrichment near the transcription start site, whereas Set2 was distributed along pre-mRNAs. A subset of transcripts showed notably high enrichment for Set1 or Set2 binding relative to RNAPII, suggesting functional posttranscriptional interactions. In particular, Set1 was strongly bound to the SET1 mRNA, Ty1 retrotransposons, and noncoding RNAs from the ribosomal DNA (rDNA) intergenic spacers, consistent with its previously reported silencing roles. Set1 lacking RNA recognition motif 2 (RRM2) showed reduced in vivo cross-linking to RNA and reduced chromatin occupancy. In addition, levels of H3K4 trimethylation were decreased, whereas levels of dimethylation were increased. We conclude that RNA binding by Set1 contributes to both chromatin association and methyltransferase activity.

scholarly journals RNA-recognition motif in Matrin-3 mediates neurodegeneration through interaction with hnRNPM

2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Nandini Ramesh ◽  
Sukhleen Kour ◽  
Eric N. Anderson ◽  
Dhivyaa Rajasundaram ◽  
Udai Bhan Pandey
Keyword(s):  
Motor Neurons ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Dependent Manner ◽  
Recognition Motif ◽  
Matrin 3

Abstract Background Amyotrophic lateral sclerosis (ALS) is an adult-onset, fatal neurodegenerative disease characterized by progressive loss of upper and lower motor neurons. While pathogenic mutations in the DNA/RNA-binding protein Matrin-3 (MATR3) are linked to ALS and distal myopathy, the molecular mechanisms underlying MATR3-mediated neuromuscular degeneration remain unclear. Methods We generated Drosophila lines with transgenic insertion of human MATR3 wildtype, disease-associated variants F115C and S85C, and deletion variants in functional domains, ΔRRM1, ΔRRM2, ΔZNF1 and ΔZNF2. We utilized genetic, behavioral and biochemical tools for comprehensive characterization of our models in vivo and in vitro. Additionally, we employed in silico approaches to find transcriptomic targets of MATR3 and hnRNPM from publicly available eCLIP datasets. Results We found that targeted expression of MATR3 in Drosophila muscles or motor neurons shorten lifespan and produces progressive motor defects, muscle degeneration and atrophy. Strikingly, deletion of its RNA-recognition motif (RRM2) mitigates MATR3 toxicity. We identified rump, the Drosophila homolog of human RNA-binding protein hnRNPM, as a modifier of mutant MATR3 toxicity in vivo. Interestingly, hnRNPM physically and functionally interacts with MATR3 in an RNA-dependent manner in mammalian cells. Furthermore, common RNA targets of MATR3 and hnRNPM converge in biological processes important for neuronal health and survival. Conclusions We propose a model of MATR3-mediated neuromuscular degeneration governed by its RNA-binding domains and modulated by interaction with splicing factor hnRNPM.

scholarly journals CUS2, a Yeast Homolog of Human Tat-SF1, Rescues Function of Misfolded U2 through an Unusual RNA Recognition Motif

1998 ◽  
Vol 18 (9) ◽  
pp. 5000-5009 ◽  
Author(s):  
Dong Yan ◽  
Rhonda Perriman ◽  
Haller Igel ◽  
Kenneth J. Howe ◽  
Megan Neville ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Normal Activity ◽  
Binding Activity ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Recognition Motif ◽  
Yeast Homolog

ABSTRACT A screen for suppressors of a U2 snRNA mutation identified CUS2, an atypical member of the RNA recognition motif (RRM) family of RNA binding proteins. CUS2 protein is associated with U2 RNA in splicing extracts and interacts with PRP11, a subunit of the conserved splicing factor SF3a. Absence of CUS2 renders certain U2 RNA folding mutants lethal, arguing that a normal activity of CUS2 is to help refold U2 into a structure favorable for its binding to SF3b and SF3a prior to spliceosome assembly. Both CUS2 function in vivo and the in vitro RNA binding activity of CUS2 are disrupted by mutation of the first RRM, suggesting that rescue of misfolded U2 involves the direct binding of CUS2. Human Tat-SF1, reported to stimulate Tat-specific, transactivating region-dependent human immunodeficiency virus transcription in vitro, is structurally similar to CUS2. Anti-Tat-SF1 antibodies coimmunoprecipitate SF3a66 (SAP62), the human homolog of PRP11, suggesting that Tat-SF1 has a parallel function in splicing in human cells.

scholarly journals Regulation of pri-miRNA processing by the hnRNP-like protein AtGRP7 in Arabidopsis

2014 ◽  
Vol 42 (15) ◽  
pp. 9925-9936 ◽  
Author(s):  
Tino Köster ◽  
Katja Meyer ◽  
Claus Weinholdt ◽  
Lisa M. Smith ◽  
Martina Lummer ◽  
...  
Keyword(s):  
Rna Binding ◽  
Mature Mirnas ◽  
Mrna Splicing ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Mirna Processing ◽  
Rna Immunoprecipitation ◽  
Direct Binding ◽  
Interfering Rna

Abstract The hnRNP-like glycine-rich RNA-binding protein AtGRP7 regulates pre-mRNA splicing in Arabidopsis. Here we used small RNA-seq to show that AtGRP7 also affects the miRNA inventory. AtGRP7 overexpression caused a significant reduction in the level of 30 miRNAs and an increase for 14 miRNAs with a minimum log2 fold change of ±0.5. Overaccumulation of several pri-miRNAs including pri-miR398b, pri-miR398c, pri-miR172b, pri-miR159a and pri-miR390 at the expense of the mature miRNAs suggested that AtGRP7 affects pri-miRNA processing. Indeed, RNA immunoprecipitation revealed that AtGRP7 interacts with these pri-miRNAs in vivo. Mutation of an arginine in the RNA recognition motif abrogated in vivo binding and the effect on miRNA and pri-miRNA levels, indicating that AtGRP7 inhibits processing of these pri-miRNAs by direct binding. In contrast, pri-miRNAs of selected miRNAs that were elevated or not changed in response to high AtGRP7 levels were not bound in vivo. Reduced accumulation of miR390, an initiator of trans-acting small interfering RNA (ta-siRNA) formation, also led to lower TAS3 ta-siRNA levels and increased mRNA expression of the target AUXIN RESPONSE FACTOR4. Furthermore, AtGRP7 affected splicing of pri-miR172b and pri-miR162a. Thus, AtGRP7 is an hnRNP-like protein with a role in processing of pri-miRNAs in addition to its role in pre-mRNA splicing.

scholarly journals Rna15 Interaction with the A-Rich Yeast Polyadenylation Signal Is an Essential Step in mRNA 3′-End Formation

2001 ◽  
Vol 21 (23) ◽  
pp. 8045-8055 ◽  
Author(s):  
Stefan Gross ◽  
Claire L. Moore
Keyword(s):  
Rna Binding ◽  
Contact Point ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Binding Motifs ◽  
Factor I ◽  
Polyadenylation Factor ◽  
Mammalian System

ABSTRACT In Saccharomyces cerevisiae, four factors [cleavage factor I (CF I), CF II, polyadenylation factor I (PF I), and poly(A) polymerase (PAP)] are required for maturation of the 3′ end of the mRNA. CF I and CF II are required for cleavage; a complex of PAP and PF I, which includes CF II subunits, participates in polyadenylation, along with CF I. These factors are directed to the appropriate site on the mRNA by two sequences: one A-rich and one UA-rich. CF I contains five proteins, two of which, Rna15 and Hrp1, interact with the mRNA through RNA recognition motif-type RNA binding motifs. Previous work demonstrated that the UV cross-linking of purified Hrp1 to RNA required the UA-rich element, but the contact point of Rna15 was not known. We show here that Rna15 does not recognize a particular sequence in the absence of other proteins. However, in complex with Hrp1 and Rna14, Rna15 specifically interacts with the A-rich element. The Pcf11 and Clp1 subunits of CF I are not needed to position Rna15 at this site. This interaction is essential to the function of CF I. A mutant Rna15 with decreased affinity for RNA is defective for in vitro RNA processing and lethal in vivo, while an RNA with a mutation in the A-rich element is not processed in vitro and can no longer be UV cross-linked to the Rna15 subunit assembled into CF I. Thus, the recognition of the A-rich element depends on the tethering of Rna15 through an Rna14 bridge to Hrp1 bound to the UA-rich motif. These results illustrate that the yeast 3′ end is defined and processed by a mechanism surprisingly different from that used by the mammalian system.

scholarly journals RNA Binding Activity of Heterodimeric Splicing Factor U2AF: at Least One RS Domain Is Required for High-Affinity Binding

1998 ◽  
Vol 18 (7) ◽  
pp. 4004-4011 ◽  
Author(s):  
David Z. Rudner ◽  
Kevin S. Breger ◽  
Roland Kanaar ◽  
Melissa D. Adams ◽  
Donald C. Rio
Keyword(s):  
Rna Binding ◽  
Large Subunit ◽  
Small Subunit ◽  
Binding Activity ◽  
Splicing Factor ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Rs Domains

ABSTRACT The pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein particle [snRNP] auxiliary factor) plays a critical role in 3′ splice site selection. U2AF binds site specifically to the intron pyrimidine tract between the branchpoint and the 3′ splice site and targets U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF65) and small (hU2AF35) subunits. hU2AF65 contains an arginine-serine-rich (RS) domain and three RNA recognition motifs (RRMs). hU2AF35 has a degenerate RRM and a carboxyl-terminal RS domain. Genetic studies have recently shown that the RS domains on the Drosophila U2AF subunit homologs are each inessential and might have redundant functions in vivo. The site-specific pyrimidine tract binding activity of the U2AF heterodimer has previously been assigned to hU2AF65. While the requirement for the three RRMs on hU2AF65 is firmly established, a role for the large-subunit RS domain in RNA binding remains unresolved. We have analyzed the RNA binding activity of the U2AF heterodimer in vitro. When theDrosophila small-subunit homolog (dU2AF38) was complexed with the large-subunit (dU2AF50) pyrimidine tract, RNA binding activity increased 20-fold over that of free dU2AF50. We detected a similar increase in RNA binding activity when we compared the human U2AF heterodimer and hU2AF65. Surprisingly, the RS domain on dU2AF38was necessary for the increased binding activity of the dU2AF heterodimer. In addition, removal of the RS domain from theDrosophila large-subunit monomer (dU2AF50ΔRS) severely impaired its binding activity. However, if the dU2AF38 RS domain was supplied in a complex with dU2AF50ΔRS, high-affinity binding was restored. These results suggest that the presence of one RS domain of U2AF, on either the large or small subunit, promotes high-affinity pyrimidine tract RNA binding activity, consistent with redundant roles for the U2AF RS domains in vivo.

scholarly journals Analysis of Mutant Phenotypes and Splicing Defects Demonstrates Functional Collaboration between the Large and Small Subunits of the Essential Splicing Factor U2AF In Vivo

2005 ◽  
Vol 16 (2) ◽  
pp. 584-596 ◽  
Author(s):  
Christopher J. Webb ◽  
Sujata Lakhe-Reddy ◽  
Charles M. Romfo ◽  
Jo Ann Wise
Keyword(s):  
Splice Site ◽  
Large Subunit ◽  
Small Subunit ◽  
Splicing Factor ◽  
In Vitro Assays ◽  
Growth Defect ◽  
Mutant Phenotypes ◽  
Site Recognition

The heterodimeric splicing factor U2AF plays an important role in 3′ splice site selection, but the division of labor between the two subunits in vivo remains unclear. In vitro assays led to the proposal that the human large subunit recognizes 3′ splice sites with extensive polypyrimidine tracts independently of the small subunit. We report in vivo analysis demonstrating that all five domains of spU2AFLGare essential for viability; a partial deletion of the linker region, which forms the small subunit interface, produces a severe growth defect and an aberrant morphology. A small subunit zinc-binding domain mutant confers a similar phenotype, suggesting that the heterodimer functions as a unit during splicing in Schizosaccharomyces pombe. As this is not predicted by the model for metazoan 3′ splice site recognition, we sought introns for which the spU2AFLGand spU2AFSMmake distinct contributions by analyzing diverse splicing events in strains harboring mutations in each partner. Requirements for the two subunits are generally parallel and, moreover, do not correlate with the length or strength of the 3′ pyrimidine tract. These and other studies performed in fission yeast support a model for 3′ splice site recognition in which the two subunits of U2AF functionally collaborate in vivo.

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