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 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.

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.

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.

scholarly journals Structure, dynamics and roX2-lncRNA binding of tandem double-stranded RNA binding domains dsRBD1,2 of Drosophila helicase Maleless

2018 ◽  
Author(s):  
Pravin Kumar Ankush Jagtap ◽  
Marisa Müller ◽  
Pawel Masiewicz ◽  
Sören von Bülow ◽  
Nele Merret Hollmann ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Motifs ◽  
Double Stranded Rna ◽  
Binding Domains ◽  
Dsrna Binding ◽  
Rna Binding Domains ◽  
Human Orthologue ◽  
Rox Rna

ABSTRACTMaleless (MLE) is an evolutionary conserved member of the DExH family of helicases in Drosophila. Besides its function in RNA editing and presumably siRNA processing, MLE is best known for its role in remodelling non-coding roX RNA in the context of X chromosome dosage compensation in male flies. MLE and its human orthologue, DHX9 contain two tandem double-stranded RNA binding domains (dsRBDs) located at the N-terminal region. The two dsRBDs are essential for localization of MLE at the X-territory and it is presumed that this involves binding roX secondary structures. However, for dsRBD1 roX RNA binding has so far not been described. Here, we determined the solution NMR structure of dsRBD1 and dsRBD2 of MLE in tandem and investigated its role in double-stranded RNA (dsRNA) binding. Our NMR data show that both dsRBDs act as independent structural modules in solution and are canonical, non-sequence-specific dsRBDs featuring non-canonical KKxAK RNA binding motifs. NMR titrations combined with filter binding experiments document the contribution of dsRBD1 to dsRNA binding in vitro. Curiously, dsRBD1 mutants in which dsRNA binding in vitro is strongly compromised do not affect roX2 RNA binding and MLE localization in cells. These data suggest alternative functions for dsRBD1 in vivo.

scholarly journals Engineered Exosomes for the Targeted Delivery of a Novel Therapeutic Cargo to Enhance Sorafenib-Mediated Ferroptosis in Hepatocellular Carcinoma.

2021 ◽  
Author(s):  
Xiaoju Li ◽  
Qianqian Yu ◽  
Xinyan Guo ◽  
Chenlin Liu ◽  
Runze Zhao ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Targeted Delivery ◽  
Rna Recognition Motif ◽  
Advanced Hepatocellular Carcinoma ◽  
Rna Recognition ◽  
Sorafenib Treatment ◽  
Recognition Motif ◽  
Interfering Rna

Abstract Background Sorafenib is one of the few effective first-line drugs approved for the treatment of advanced hepatocellular carcinoma (HCC). However, the development of drug resistance is common among individuals with HCC. Thus, there is an urgent need to solve this problem. Results Recent evidence indicated that the anticancer activity of sorafenib mainly relies on the induction of ferroptosis. In our study, genes that suppress ferroptosis, especially GPX4 and DHODH, were enriched in sorafenib-resistant cells and primary tissues and were associated with poor prognosis of HCC patients who received sorafenib treatment. Therefore, silencing GPX4 and DHODH might be a novel and effective strategy to overcome sorafenib resistance. Here, a novel ferroptosis inducer comprising a multiplex small interfering RNA (multi-siRNA) capable of simultaneously silencing GPX4 and DHODH was created. Then, exosomes with high multi-siRNA loading and HCC-specific targeting were established by fusing the SP94 peptide and the N-terminal RNA recognition motif (RRM) of U1-A with the exosomal membrane protein Lamp2b. The results from the in vitro and in vivo experiments indicate that this tumor-targeting nanodelivery system (ExoSP94−lamp2b−RRM-multi-siRNA) could enhance sorafenib-induced ferroptosis and overcome sorafenib resistance, which might open a new avenue for clinically overcoming sorafenib resistance. Conclusions We designed HCC-targeted exosomes (ExoSP94−Lamp2b−RRM) that can deliver a novel ferroptosis inducer. Our data show that ExoSP94−lamp2b−RRM-multi-siRNA could enhance sorafenib-induced ferroptosis by silencing GPX4 and DHODH expression and consequently increase HCC sensitivity to sorafenib. This is the first study to describe the use of engineered exosomes to overcome acquired sorafenib resistance with respect to ferroptosis.

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 Multifunctional G-Rich and RRM-Containing Domains of TbRGG2 Perform Separate yet Essential Functions in Trypanosome RNA Editing

2012 ◽  
Vol 11 (9) ◽  
pp. 1119-1131 ◽  
Author(s):  
Bardees M. Foda ◽  
Kurtis M. Downey ◽  
John C. Fisk ◽  
Laurie K. Read
Keyword(s):  
Rna Editing ◽  
Rna Binding ◽  
Rna Recognition Motif ◽  
Rich Region ◽  
Content Type ◽  
High Affinity ◽  
Rich Domain ◽  
Rrm Domain

ABSTRACT Efficient editing of Trypanosoma brucei mitochondrial RNAs involves the actions of multiple accessory factors. T. brucei RGG2 (TbRGG2) is an essential protein crucial for initiation and 3′-to-5′ progression of editing. TbRGG2 comprises an N-terminal G-rich region containing GWG and RG repeats and a C-terminal RNA recognition motif (RRM)-containing domain. Here, we perform in vitro and in vivo separation-of-function studies to interrogate the mechanism of TbRGG2 action in RNA editing. TbRGG2 preferentially binds preedited mRNA in vitro with high affinity attributable to its G-rich region. RNA-annealing and -melting activities are separable, carried out primarily by the G-rich and RRM domains, respectively. In vivo , the G-rich domain partially complements TbRGG2 knockdown, but the RRM domain is also required. Notably, TbRGG2's RNA-melting activity is dispensable for RNA editing in vivo . Interactions between TbRGG2 and MRB1 complex proteins are mediated by both G-rich and RRM-containing domains, depending on the binding partner. Overall, our results are consistent with a model in which the high-affinity RNA binding and RNA-annealing activities of the G-rich domain are essential for RNA editing in vivo . The RRM domain may have key functions involving interactions with the MRB1 complex and/or regulation of the activities of the G-rich domain.

scholarly journals Bioassaying Putative RNA-Binding Motifs in a Protein Encoded by a Gene That Influences Courtship and Visually Mediated Behavior in Drosophila: In Vitro Mutagenesis of nonA

Genetics ◽  
1996 ◽  
Vol 143 (1) ◽  
pp. 259-275
Author(s):  
Ralf Stanewsky ◽  
Thomas A Fry ◽  
Ingolf Reim ◽  
Harald Saumwebert ◽  
Jeffrey C Hall
Keyword(s):  
Rna Binding ◽  
Courtship Song ◽  
In Vitro Mutagenesis ◽  
Rna Recognition ◽  
Null Mutant ◽  
Rna Recognition Motifs ◽  
Binding Motifs ◽  
Recognition Motifs ◽  
Transgenic Strains

Abstract The no-on-transient-A (nonA) gene of Drosophila melanogaster influences vision, courtship song, and viability. The nod-encoded polypeptide is inferred to bind single-stranded nucleic acids. Although sequence-analysis of NONA implies that it belongs to a special interspecific family of this protein type, it does contain two classical RNA recognition motifs (RRM). Their behavioral significance was assayed by generating transgenic strains that were singly or multiply mutated within the relatively N-terminal motif (RRM1) or within RRM2. Neither class of mutation affected NONA binding to polytene chromcsomes. The former mutations led to extremely low viability, accompanied by diminished adult longevities that were much worse than for a nod-null mutant, implying that faulty interpolypeptide interactions might accompany the effects of the amino-acid substitutions within RRM1. All in vitro-mutated types caused optomotor blindness and an absence of transient spikes in the electroretinogram. Courtship analysis discriminated between the effects of the mutations: the RRM2-mutated type generated song pulses and trains that tended to be mildly mutant. These phenotypic abnormalities reinforce the notion that nonA' s ubiquitous expression has its most important consequences in the optic lobes, the thoracic ganglia, or both, depending in part on the nonA allele.

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