Structural analysis of the C-terminal domain of the spliceosomal helicase Prp22

2012 ◽  
Vol 393 (10) ◽  
pp. 1131-1140 ◽  
Author(s):  
Denis Kudlinzki ◽  
Andreas Schmitt ◽  
Henning Christian ◽  
Ralf Ficner
Keyword(s):  
Rna Binding ◽  
Dna Helicase ◽  
Specific Interactions ◽  
Winged Helix ◽  
Dsrna Binding ◽  
Binding Surface ◽  
Rna Complexes ◽  
Positively Charged ◽  
Analogous Function

Abstract Splicing of pre-mRNA requires the activity of at least eight different DEAD/H-box proteins that are involved in distinct steps of the splicing process. These proteins are driving the spliceosomal machinery by ATP-dependent unwinding of dsRNA and/or disrupting protein-RNA complexes. The spliceosomal DEAH-box proteins Prp2, Prp16, Prp22 and Prp43 share homologous C-terminal domains (CTD). We have determined the crystal structure of the CTD of human Prp22 by means of MAD. The fold of the human Prp22-CTD closely resembles that of the yeast Prp43-CTD. The similarity of these helicase-associated CTDs to the winged-helix and ratchet domains of the DNA helicase Hel308 suggests an analogous function in dsRNA binding and unwinding. Here, we also demonstrate that the CTD has a significant impact on the ATPase activity of yPrp22 in vitro. Homology modeling of the CTDs of hPrp2, hPrp16 and hPrp43 suggests that the CTDs of spliceosomal helicases contain conserved positively charged patches on their surfaces representing a common RNA-binding surface as well as divergent regions most likely mediating specific interactions with different proteins of the spliceosome.

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 Trafficking of siRNA precursors by the dsRBD protein Blanks in Drosophila

2020 ◽  
Vol 48 (7) ◽  
pp. 3906-3921 ◽  
Author(s):  
Volker Nitschko ◽  
Stefan Kunzelmann ◽  
Thomas Fröhlich ◽  
Georg J Arnold ◽  
Klaus Förstemann
Keyword(s):  
Small Rnas ◽  
Rna Binding ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Binding Domains ◽  
Convergent Transcription ◽  
Dsrna Binding ◽  
Functional Screens

Abstract RNA interference targets aberrant transcripts with cognate small interfering RNAs, which derive from double-stranded RNA precursors. Several functional screens have identified Drosophila blanks/lump (CG10630) as a facilitator of RNAi, yet its molecular function has remained unknown. The protein carries two dsRNA binding domains (dsRBD) and blanks mutant males have a spermatogenesis defect. We demonstrate that blanks selectively boosts RNAi triggered by dsRNA of nuclear origin. Blanks binds dsRNA via its second dsRBD in vitro, shuttles between nucleus and cytoplasm and the abundance of siRNAs arising at many sites of convergent transcription is reduced in blanks mutants. Since features of nascent RNAs - such as introns and transcription beyond the polyA site – contribute to the small RNA pool, we propose that Blanks binds dsRNA formed by cognate nascent RNAs in the nucleus and fosters its export to the cytoplasm for dicing. We refer to the resulting small RNAs as blanks exported siRNAs (bepsiRNAs). While bepsiRNAs were fully dependent on RNA binding to the second dsRBD of blanks in transgenic flies, male fertility was not. This is consistent with a previous report that linked fertility to the first dsRBD of Blanks. The role of blanks in spermatogenesis appears thus unrelated to its role in dsRNA export.

Abstract 257: Selective Targeting of a Novel Epsin-VEGFR2 Interaction Promotes VEGF-mediated Angiogenesis

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
H N Rahman ◽  
Hao Wu ◽  
Satish Pasula ◽  
Yunzhou Dong ◽  
Aiyun Wen ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Endothelial Cell Proliferation ◽  
Specific Interactions ◽  
Vegf Signaling ◽  
Binding Surface ◽  
Selective Targeting ◽  
Molecular Determinants ◽  
Retinal Angiogenesis

Background: VEGF-induced binding of VEGFR2 to epsins 1 and 2 triggers VEGFR2 degradation and attenuates VEGF signaling. The epsin ubiquitin interacting motif (UIM) was shown to be required for the interaction with VEGFR2, however the molecular determinants that govern how epsin specifically interacts with and regulates VEGFR2 were unknown. The goals for the present study were (1) to identify critical molecular determinants that drive the specificity of the epsin and VEGFR2 interaction and (2) to ascertain if such determinants were critical for physiological angiogenesis in vivo . Methods and Results: Structural modeling uncovered two novel binding surfaces within VEGFR2 that mediate specific interactions with epsin UIM. Three glutamic acid residues in epsin UIM were found to interact with residues in VEGFR2. Further, we found that the VEGF-induced VEGFR2-epsin interaction promoted c-Cbl-mediated ubiquitination of epsin, and uncovered a previously unappreciated ubiquitin-binding surface within VEGFR2. Mutational analysis revealed that the VEGFR2-epsin interaction is supported by VEGFR2 interacting specifically with the UIM and with ubiquitinated epsin. An epsin UIM peptide, but not a mutant UIM peptide, potentiated endothelial cell proliferation, migration and angiogenic properties in vitro , increased postnatal retinal angiogenesis, and enhanced VEGF-induced physiological angiogenesis and wound healing. Conclusions: Distinct residues in the epsin UIM and VEGFR2 mediate specific interactions between epsin and VEGFR2, in addition to UIM recognition of ubiquitin moieties on VEGFR2. These novel interactions are critical for pathophysiological angiogenesis, suggesting that these sites could be selectively targeted by therapeutics to modulate angiogenesis.

scholarly journals Dissection of Double-Stranded RNA Binding Protein B2 from Betanodavirus

2007 ◽  
Vol 81 (11) ◽  
pp. 5449-5459 ◽  
Author(s):  
Beau J. Fenner ◽  
Winnie Goh ◽  
Jimmy Kwang
Keyword(s):  
Rna Silencing ◽  
Rna Binding ◽  
Amino Acid Residues ◽  
Alanine Scanning Mutagenesis ◽  
Marine Aquaculture ◽  
Double Stranded Rna ◽  
Dsrna Binding ◽  
Fish Cells ◽  
Interfering Rna

ABSTRACT Betanodaviruses are small RNA viruses that infect teleost fish and pose a considerable threat to marine aquaculture production. These viruses possess a small protein, termed B2, which binds to and protects double-stranded RNA. This prevents cleavage of virus-derived double-stranded RNAs (dsRNAs) by Dicer and subsequent production of small interfering RNA (siRNA), which would otherwise induce an RNA-silencing response against the virus. In this work, we have performed charged-to-alanine scanning mutagenesis of the B2 protein in order to identify residues required for dsRNA binding and protection. While the majority of the 19 mutated B2 residues were required for maximal dsRNA binding and protection in vitro, residues R53 and R60 were essential for both activities. Subsequent experiments in fish cells confirmed these findings by showing that mutations in these residues abolished accumulation of both the RNA1 and RNA2 components of the viral genome, in addition to preventing any significant induction of the host interferon gene, Mx. Moreover, an obvious positive correlation was found between dsRNA binding and protection in vitro and RNA1, RNA2, and Mx accumulation in fish cells, further validating the importance of the selected amino acid residues. The same trend was also demonstrated using an RNA silencing system in HeLa cells, with residues R53 and R60 being essential for suppression of RNA silencing. Importantly, we found that siRNA-mediated knockdown of Dicer dramatically enhanced the accumulation of a B2 mutant. In addition, we found that B2 is able to induce apoptosis in fish cells but that this was not the result of dsRNA binding.

scholarly journals A Human Sequence Homologue of Staufen Is an RNA-Binding Protein That Is Associated with Polysomes and Localizes to the Rough Endoplasmic Reticulum

1999 ◽  
Vol 19 (3) ◽  
pp. 2212-2219 ◽  
Author(s):  
Rosa María Marión ◽  
Puri Fortes ◽  
Ana Beloso ◽  
Carlos Dotti ◽  
Juan Ortín
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Rna Binding ◽  
Intracellular Localization ◽  
Binding Activity ◽  
Ns1 Protein ◽  
Binding Domains ◽  
Human Sequence ◽  
Dsrna Binding

ABSTRACT In the course of a two-hybrid screen with the NS1 protein of influenza virus, a human clone capable of coding for a protein with high homology to the Staufen protein from Drosophila melanogaster (dmStaufen) was identified. With these sequences used as a probe, cDNAs were isolated from a λ cDNA library. The encoded protein (hStaufen-like) contained four double-stranded RNA (dsRNA)-binding domains with 55% similarity and 38% identity to those of dmStaufen, including identity at all residues involved in RNA binding. A recombinant protein containing all dsRNA-binding domains was expressed in Escherichia coli as a His-tagged polypeptide. It showed dsRNA binding activity in vitro, with an apparentKd of 10−9 M. Using a specific antibody, we detected in human cells a major form of the hStaufen-like protein with an apparent molecular mass of 60 to 65 kDa. The intracellular localization of hStaufen-like protein was investigated by immunofluorescence using a series of markers for the cell compartments. Colocalization was observed with the rough endoplasmic reticulum but not with endosomes, cytoskeleton, or Golgi apparatus. Furthermore, sedimentation analyses indicated that hStaufen-like protein associates with polysomes. These results are discussed in relation to the possible functions of the protein.

scholarly journals A positively charged surface patch on the pestivirus NS3 protease module plays an important role in modulating NS3 helicase activity and virus production

2021 ◽  
Author(s):  
Fengwei Zheng ◽  
Weicheng Yi ◽  
Weichi Liu ◽  
Hongchang Zhu ◽  
Peng Gong ◽  
...  
Keyword(s):  
Rna Binding ◽  
Nonstructural Protein ◽  
Virus Production ◽  
Classical Swine Fever ◽  
Surface Patch ◽  
Helicase Activity ◽  
Residue Substitution ◽  
Ns3 Protease ◽  
Positively Charged

Abstract Pestivirus nonstructural protein 3 (NS3) is a multifunctional protein with protease and helicase activities that are essential for the virus replication. In this study, we used a combination of biochemical and genetic approaches to investigate the relationship between the positively charged patch on protease module and NS3 function. The surface patch was composed of four basic residues R50, K74 and K94 in NS3 protease domain and H24 in the structurally integrated cofactor NS4A PCS . Single residue or simultaneous four-residue substitution in the patch to alanine or aspartic acid hardly affect ATPase activity. However, the single R50, K94 or H24 residue or simultaneous four-residue substitution resulted in the apparent changes of the helicase activity and RNA-binding ability of NS3. When these mutations were introduced into a classical swine fever virus (CSFV) cDNA clone, the single K94 residue or simultaneous four-residue substitution (Qua_A or Qua_D) impaired the infectious virus production. Furthermore, the replication efficiency of the CSFV variants was partially correlated to the helicase activity of NS3 in vitro . Our results suggest that the conserved positively charged patch on the NS3 plays an important role in modulating the NS3 helicase activity in vitro and CSFV production.

scholarly journals Architecture and RNA binding of the human negative elongation factor

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Seychelle M Vos ◽  
David Pöllmann ◽  
Livia Caizzi ◽  
Katharina B Hofmann ◽  
Pascaline Rombaut ◽  
...  
Keyword(s):  
Rna Binding ◽  
Three Dimensional ◽  
Elongation Factor ◽  
X Ray Crystallography ◽  
Biochemical Assays ◽  
Opposite Face ◽  
Negative Elongation Factor ◽  
Positively Charged

Transcription regulation in metazoans often involves promoter-proximal pausing of RNA polymerase (Pol) II, which requires the 4-subunit negative elongation factor (NELF). Here we discern the functional architecture of human NELF through X-ray crystallography, protein crosslinking, biochemical assays, and RNA crosslinking in cells. We identify a NELF core subcomplex formed by conserved regions in subunits NELF-A and NELF-C, and resolve its crystal structure. The NELF-AC subcomplex binds single-stranded nucleic acids in vitro, and NELF-C associates with RNA in vivo. A positively charged face of NELF-AC is involved in RNA binding, whereas the opposite face of the NELF-AC subcomplex binds NELF-B. NELF-B is predicted to form a HEAT repeat fold, also binds RNA in vivo, and anchors the subunit NELF-E, which is confirmed to bind RNA in vivo. These results reveal the three-dimensional architecture and three RNA-binding faces of NELF.

scholarly journals Defined d-hexapeptides bind CUG repeats and rescue phenotypes of myotonic dystrophy myotubes in a Drosophila model of the disease

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anna Rapisarda ◽  
Ariadna Bargiela ◽  
Beatriz Llamusi ◽  
Isabel Pont ◽  
Roger Estrada-Tejedor ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Ctg Repeats ◽  
Rna Hairpins ◽  
Drosophila Model ◽  
Toxic Rna ◽  
Rna Complexes ◽  
Cug Repeats

AbstractIn Myotonic Dystrophy type 1 (DM1), a non-coding CTG repeats rare expansion disease; toxic double-stranded RNA hairpins sequester the RNA-binding proteins Muscleblind-like 1 and 2 (MBNL1 and 2) and trigger other DM1-related pathogenesis pathway defects. In this paper, we characterize four d-amino acid hexapeptides identified together with abp1, a peptide previously shown to stabilize CUG RNA in its single-stranded conformation. With the generalized sequence cpy(a/t)(q/w)e, these related peptides improved three MBNL-regulated exon inclusions in DM1-derived cells. Subsequent experiments showed that these compounds generally increased the relative expression of MBNL1 and its nuclear-cytoplasmic distribution, reduced hyperactivated autophagy, and increased the percentage of differentiated (Desmin-positive) cells in vitro. All peptides rescued atrophy of indirect flight muscles in a Drosophila model of the disease, and partially rescued muscle function according to climbing and flight tests. Investigation of their mechanism of action supports that all four compounds can bind to CUG repeats with slightly different association constant, but binding did not strongly influence the secondary structure of the toxic RNA in contrast to abp1. Finally, molecular modeling suggests a detailed view of the interactions of peptide-CUG RNA complexes useful in the chemical optimization of compounds.

Abstract 304: Selective Targeting of a Novel Epsin-VEGFR2 Interaction Promotes VEGF-mediated Angiogenesis

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Hong Chen
Keyword(s):  
Mutational Analysis ◽  
Endothelial Cell Proliferation ◽  
Specific Interactions ◽  
Vegf Signaling ◽  
Binding Surface ◽  
Selective Targeting ◽  
Molecular Determinants ◽  
Retinal Angiogenesis

Background: VEGF-induced binding of VEGFR2 to epsins 1 and 2 triggers VEGFR2 degradation and attenuates VEGF signaling. The epsin ubiquitin interacting motif (UIM) was shown to be required for the interaction with VEGFR2, however the molecular determinants that govern how epsin specifically interacts with and regulates VEGFR2 were unknown. The goals for the present study were (1) to identify critical molecular determinants that drive the specificity of the epsin and VEGFR2 interaction and (2) to ascertain if such determinants were critical for physiological angiogenesis in vivo . Methods and Results: Structural modeling uncovered two novel binding surfaces within VEGFR2 that mediate specific interactions with epsin UIM. Three glutamic acid residues in epsin UIM were found to interact with residues in VEGFR2. Further, we found that the VEGF-induced VEGFR2-epsin interaction promoted c-Cbl-mediated ubiquitination of epsin, and uncovered a previously unappreciated ubiquitin-binding surface within VEGFR2. Mutational analysis revealed that the VEGFR2-epsin interaction is supported by VEGFR2 interacting specifically with the UIM and with ubiquitinated epsin. An epsin UIM peptide, but not a mutant UIM peptide, potentiated endothelial cell proliferation, migration and angiogenic properties in vitro , increased postnatal retinal angiogenesis, and enhanced VEGF-induced physiological angiogenesis and wound healing. Conclusions: Distinct residues in the epsin UIM and VEGFR2 mediate specific interactions between epsin and VEGFR2, in addition to UIM recognition of ubiquitin moieties on VEGFR2. These novel interactions are critical for pathophysiological angiogenesis, suggesting that these sites could be selectively targeted by therapeutics to modulate angiogenesis.

scholarly journals RNA-binding properties of the 63 kDa protein encoded by the triple gene block of poa semilatent hordeivirus

2001 ◽  
Vol 82 (10) ◽  
pp. 2569-2578 ◽  
Author(s):  
N. O. Kalinina ◽  
D. A. Rakitina ◽  
N. E. Yelina ◽  
A. A. Zamyatnin ◽  
T. A. Stroganova ◽  
...  
Keyword(s):  
Rna Binding ◽  
Triple Gene Block ◽  
Helicase Domain ◽  
Sequence Motifs ◽  
Long Distance ◽  
Binding Properties ◽  
Gene Block ◽  
Extension Domain ◽  
Positively Charged

The 63 kDa ‘63K’ movement protein encoded by the triple gene block of poa semilatent virus (PSLV) comprises the C-terminal NTPase/helicase domain and the N-terminal extension domain, which contains two positively charged sequence motifs, A and B. In this study, the in vitro RNA-binding properties of PSLV 63K and its mutants were analysed. Membrane-immobilized 63K and N-63K (isolated N-terminal extension domain) bound RNA at high NaCl concentrations. In contrast, C-63K (isolated NTPase/helicase domain) was able to bind RNA only at NaCl concentrations of up to 50 mM. In gel-shift assays, C-63K bound RNA to form complexes that were unable to enter an agarose gel, whereas complexes formed by N-63K could enter the gel. Full-length 63K formed both types of complexes. Visualization of the RNA–protein complexes formed by 63K, N-63K and C-63K by atomic force microscopy demonstrated that each complex had a different shape. Collectively, these data indicate that 63K has two distinct RNA-binding activities associated with the NTPase/helicase domain and the N-terminal extension domain. Mutations in either of the positively charged sequence motifs A and B had little effect on the RNA binding of the N-terminal extension domain, whereas mutations in both motifs together inhibited RNA binding. Hybrid viruses with mutations in motifs A and B were able to infect inoculated leaves of Nicotiana benthamiana plants, but were unable to move systemically to uninoculated leaves, suggesting that the RNA-binding activity of the N-terminal extension domain of PSLV 63K is associated with virus long-distance movement.

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