scholarly journals Functional Dissection of the Human TNRC6 (GW182-Related) Family of Proteins

2009 ◽  
Vol 29 (15) ◽  
pp. 4144-4155 ◽  
Author(s):  
David Baillat ◽  
Ramin Shiekhattar
Keyword(s):  
Mammalian Cells ◽  
Trinucleotide Repeat ◽  
Critical Role ◽  
Functional Characterization ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Critical Function ◽  
Recognition Motif ◽  
Related Family

ABSTRACT Argonaute (Ago) proteins through their association with small RNAs perform a critical function in the effector step of RNA interference. The TNRC6 (trinucleotide repeat containing 6) family of proteins have been shown to stably associate with Agos in mammalian cells. Here, we describe the isolation and functional characterization of TNRC6B- and TNRC6C-containing complexes. We show that TNRC6B and TNRC6C proteins associate with all four human Agos which are already loaded with microRNAs. Detailed domain analysis of TNRC6B protein indicated that distinct domains of the protein are required for Ago binding and P-body localization. Functional analysis using reporter constructs responsive to TNRC6B tethered through an MS2-binding domain indicates that neither the Ago-binding nor the P-body localization domains are required for translational silencing. In contrast, the C-terminal domain containing the RNA recognition motif plays a critical role in the silencing mediated by the TNRC6B protein.

scholarly journals Null Mutation of AtCUL1 Causes Arrest in Early Embryogenesis in Arabidopsis

2002 ◽  
Vol 13 (6) ◽  
pp. 1916-1928 ◽  
Author(s):  
Wen-Hui Shen ◽  
Yves Parmentier ◽  
Hanjo Hellmann ◽  
Esther Lechner ◽  
Aiwu Dong ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Critical Role ◽  
Functional Characterization ◽  
Early Embryogenesis ◽  
Cell Cycle Regulators ◽  
Genetic Studies ◽  
Scf Ubiquitin Ligase ◽  
F Box Protein

The SCF (for SKP1, Cullin/CDC53,F-box protein) ubiquitin ligase targets a number of cell cycle regulators, transcription factors, and other proteins for degradation in yeast and mammalian cells. Recent genetic studies demonstrate that plant F-box proteins are involved in auxin responses, jasmonate signaling, flower morphogenesis, photocontrol of circadian clocks, and leaf senescence, implying a large spectrum of functions for the SCF pathway in plant development. Here, we present a molecular and functional characterization of plant cullins. TheArabidopsis genome contains 11 cullin-related genes. Complementation assays revealed that AtCUL1 but not AtCUL4 can functionally complement the yeast cdc53 mutant.Arabidopsis mutants containing transfer DNA (T-DNA) insertions in the AtCUL1 gene were shown to display an arrest in early embryogenesis. Consistently, both the transcript and the protein of the AtCUL1 gene were found to accumulate in embryos. The AtCUL1 protein localized mainly in the nucleus but also weakly in the cytoplasm during interphase and colocalized with the mitotic spindle in metaphase. Our results demonstrate a critical role for the SCF ubiquitin ligase inArabidopsis embryogenesis.

scholarly journals Repression of gene expression by an exogenous sequence element acting in concert with a heterogeneous nuclear ribonucleoprotein-like protein, Nrd1, and the putative helicase Sen1.

1996 ◽  
Vol 16 (12) ◽  
pp. 6993-7003 ◽  
Author(s):  
E J Steinmetz ◽  
D A Brow
Keyword(s):  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Sequence Element ◽  
Cis Element ◽  
Recognition Motif ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Negative Effect ◽  
Nuclear Ribonucleoprotein

We have fortuitously identified a nucleotide sequence that decreases expression of a reporter gene in the yeast Saccharomyces cerevisiae 20-fold when inserted into an intron. The primary effect of the insertion is a decrease in pre-mRNA abundance accompanied by the appearance of 3'-truncated transcripts, consistent with premature transcriptional termination and/or pre-mRNA degradation. Point mutations in the cis element relieve the negative effect, demonstrating its sequence specificity. A novel yeast protein, named Nrd1, and a previously identified putative helicase, Sen1, help mediate the negative effect of the cis element. Sen1 is an essential nuclear protein that has been implicated in a variety of nuclear functions. Nrd1 has hallmarks of a heterogeneous nuclear ribonucleoprotein, including an RNA recognition motif, a region rich in RE and RS dipeptides, and a proline- and glutamine-rich domain. An N-terminal domain of Nrd1 may facilitate direct interaction with RNA polymerase II. Disruption of the NRD1 gene is lethal, yet C-terminal truncations that delete the RNA recognition motif and abrogate the negative effect of the cis element nevertheless support cell growth. Thus, expression of a gene containing the cis element could be regulated through modulation of the activity of Nrd1. The recent identification of Nrd1-related proteins in mammalian cells suggests that this potential regulatory pathway is widespread among eukaryotes.

Structural Characterization of RNA Recognition Motif-2 Domain of SART3

2017 ◽  
Vol 38 (4) ◽  
pp. 444-447
Author(s):  
Kyeong-Mi Bang ◽  
Na Youn Cho ◽  
Won-Je Kim ◽  
Ae-Ryung Kim ◽  
Hyun Kyu Song ◽  
...  
Keyword(s):  
Structural Characterization ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Recognition Motif

scholarly journals Affinity and Structural Analysis of the U1A RNA Recognition Motif with Engineered Methionines to Improve Experimental Phasing

Crystals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 273
Author(s):  
Yoshita Srivastava ◽  
Rachel Bonn-Breach ◽  
Sai Shashank Chavali ◽  
Geoffrey M. Lippa ◽  
Jermaine L. Jenkins ◽  
...  
Keyword(s):  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Molecular Replacement ◽  
Hydrophobic Core ◽  
Anomalous Diffraction ◽  
Recognition Motif ◽  
Determination Process ◽  
Experimental Phasing ◽  
Crystal Contacts ◽  
Apical Loop

RNA plays a central role in all organisms and can fold into complex structures to orchestrate function. Visualization of such structures often requires crystallization, which can be a bottleneck in the structure-determination process. To promote crystallization, an RNA-recognition motif (RRM) of the U1A spliceosomal protein has been co-opted as a crystallization module. Specifically, the U1-snRNA hairpin II (hpII) single-stranded loop recognized by U1A can be transplanted into an RNA target to promote crystal contacts and to attain phase information via molecular replacement or anomalous diffraction methods using selenomethionine. Herein, we produced the F37M/F77M mutant of U1A to augment the phasing capability of this powerful crystallization module. Selenomethionine-substituted U1A(F37M/F77M) retains high affinity for hpII (KD of 59.7 ± 11.4 nM). The 2.20 Å resolution crystal structure reveals that the mutated sidechains make new S-π interactions in the hydrophobic core and are useful for single-wavelength anomalous diffraction. Crystals were also attained of U1A(F37M/F77M) in complex with a bacterial preQ1-II riboswitch. The F34M/F37M/F77M mutant was introduced similarly into a lab-evolved U1A variant (TBP6.9) that recognizes the internal bulged loop of HIV-1 TAR RNA. We envision that this short RNA sequence can be placed into non-essential duplex regions to promote crystallization and phasing of target RNAs. We show that selenomethionine-substituted TBP6.9(F34M/F37M/F77M) binds a TAR variant wherein the apical loop was replaced with a GNRA tetraloop (KD of 69.8 ± 2.9 nM), laying the groundwork for use of TBP6.9(F34M/F37M/F77M) as a crystallization module. These new tools are available to the research community.

Identification and expression analysis of Dazl homologue in Cynops cyanurus

Zygote ◽  
2021 ◽  
pp. 1-6
Author(s):  
Yinjiao Zhao ◽  
Ya Du ◽  
Qinglan Ge ◽  
Fang Yan ◽  
Shu Wei
Keyword(s):  
Phylogenetic Analysis ◽  
Comparative Studies ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Specific Expression ◽  
Recognition Motif ◽  
Deleted In Azoospermia ◽  
Specific Expression Pattern

Summary The Dazl (deleted in azoospermia-like) gene encodes an RNA-binding protein containing an RNA recognition motif (RRM) and a DAZ motif. Dazl is essential for gametogenesis in vertebrates. In this study, we report the cloning of Dazl cDNA from Cynops cyanurus. Ccdazl mRNA showed a germline-specific expression pattern as expected. Ccdazl expression gradually decreased during oogenesis, suggesting that it may be involved in oocyte development. Phylogenetic analysis revealed that the Ccdazl protein shares conserved motifs/domains with Dazl proteins from other species. Cloning of Ccdazl provides a new tool to carry out comparative studies of germ cell development in amphibians.

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