scholarly journals Separable roles in vivo for the two RNA binding domains of a Drosophila A1-hnRNP homolog

RNA ◽  
1998 ◽  
Vol 4 (12) ◽  
pp. 1585-1598 ◽  
Author(s):  
KAI ZU ◽  
MARTHA L. SIKES ◽  
ANN L. BEYER
Keyword(s):  
Rna Binding ◽  
Binding Domains ◽  
Rna Binding Domains

scholarly journals Organizational analysis of elav gene and functional analysis of ELAV protein of Drosophila melanogaster and Drosophila virilis.

1991 ◽  
Vol 11 (6) ◽  
pp. 2994-3000 ◽  
Author(s):  
K M Yao ◽  
K White
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rna Binding ◽  
Terminal Region ◽  
Drosophila Virilis ◽  
Functional Domain ◽  
Amino Terminal ◽  
Binding Domains ◽  
Rna Binding Domains

Drosophila virilis genomic DNA corresponding to the D. melanogaster embryonic lethal abnormal visual system (elav) locus was cloned. DNA sequence analysis of a 3.8-kb genomic piece allowed identification of (i) an open reading frame (ORF) with striking homology to the previously identified D. melanogaster ORF and (ii) conserved sequence elements of possible regulatory relevance within and flanking the second intron. Conceptual translation of the D. virilis ORF predicts a 519-amino-acid-long ribonucleoprotein consensus sequence-type protein. Similar to D. melanogaster ELAV protein, it contains three tandem RNA-binding domains and an alanine/glutamine-rich amino-terminal region. The sequence throughout the RNA-binding domains, comprising the carboxy-terminal 346 amino acids, shows an extraordinary 100% identity at the amino acid level, indicating a strong structural constraint for this functional domain. The amino-terminal region is 36 amino acids longer in D. virilis, and the conservation is 66%. In in vivo functional tests, the D. virilis ORF was indistinguishable from the D. melanogaster ORF. Furthermore, a D. melanogaster ORF encoding an ELAV protein with a 40-amino-acid deletion within the alanine/glutamine-rich region was also able to supply elav function in vivo. Thus, the divergence of the amino-terminal region of the ELAV protein reflects lowered functional constraint rather than species-specific functional specification.

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 Distinct in vivo roles for double-stranded RNA-binding domains of the Xenopus RNA-editing enzyme ADAR1 in chromosomal targeting

2003 ◽  
Vol 161 (2) ◽  
pp. 309-319 ◽  
Author(s):  
Michael Doyle ◽  
Michael F. Jantsch
Keyword(s):  
Rna Editing ◽  
Rna Binding ◽  
Double Stranded Rna ◽  
Binding Domains ◽  
Initial Substrate ◽  
Rna Binding Domains ◽  
Editing Enzyme ◽  
Deaminase Domain

The RNA-editing enzyme adenosine deaminase that acts on RNA (ADAR1) deaminates adenosines to inosines in double-stranded RNA substrates. Currently, it is not clear how the enzyme targets and discriminates different substrates in vivo. However, it has been shown that the deaminase domain plays an important role in distinguishing various adenosines within a given substrate RNA in vitro. Previously, we could show that Xenopus ADAR1 is associated with nascent transcripts on transcriptionally active lampbrush chromosomes, indicating that initial substrate binding and possibly editing itself occurs cotranscriptionally. Here, we demonstrate that chromosomal association depends solely on the three double-stranded RNA-binding domains (dsRBDs) found in the central part of ADAR1, but not on the Z-DNA–binding domain in the NH2 terminus nor the catalytic deaminase domain in the COOH terminus of the protein. Most importantly, we show that individual dsRBDs are capable of recognizing different chromosomal sites in an apparently specific manner. Thus, our results not only prove the requirement of dsRBDs for chromosomal targeting, but also show that individual dsRBDs have distinct in vivo localization capabilities that may be important for initial substrate recognition and subsequent editing specificity.

Organizational analysis of elav gene and functional analysis of ELAV protein of Drosophila melanogaster and Drosophila virilis

1991 ◽  
Vol 11 (6) ◽  
pp. 2994-3000
Author(s):  
K M Yao ◽  
K White
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rna Binding ◽  
Terminal Region ◽  
Drosophila Virilis ◽  
Functional Domain ◽  
Amino Terminal ◽  
Binding Domains ◽  
Rna Binding Domains

Drosophila virilis genomic DNA corresponding to the D. melanogaster embryonic lethal abnormal visual system (elav) locus was cloned. DNA sequence analysis of a 3.8-kb genomic piece allowed identification of (i) an open reading frame (ORF) with striking homology to the previously identified D. melanogaster ORF and (ii) conserved sequence elements of possible regulatory relevance within and flanking the second intron. Conceptual translation of the D. virilis ORF predicts a 519-amino-acid-long ribonucleoprotein consensus sequence-type protein. Similar to D. melanogaster ELAV protein, it contains three tandem RNA-binding domains and an alanine/glutamine-rich amino-terminal region. The sequence throughout the RNA-binding domains, comprising the carboxy-terminal 346 amino acids, shows an extraordinary 100% identity at the amino acid level, indicating a strong structural constraint for this functional domain. The amino-terminal region is 36 amino acids longer in D. virilis, and the conservation is 66%. In in vivo functional tests, the D. virilis ORF was indistinguishable from the D. melanogaster ORF. Furthermore, a D. melanogaster ORF encoding an ELAV protein with a 40-amino-acid deletion within the alanine/glutamine-rich region was also able to supply elav function in vivo. Thus, the divergence of the amino-terminal region of the ELAV protein reflects lowered functional constraint rather than species-specific functional specification.

scholarly journals Intact RNA-binding Domains Are Necessary for Structure-specific DNA Binding and Transcription Control by CBTF122duringXenopusDevelopment

2004 ◽  
Vol 279 (50) ◽  
pp. 52447-52455 ◽  
Author(s):  
Garry P. Scarlett ◽  
Stuart J. Elgar ◽  
Peter D. Cary ◽  
Anna M. Noble ◽  
Robert L. Orford ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Rna Binding ◽  
Transcription Activator ◽  
Double Stranded Rna ◽  
Binding Domains ◽  
Rna Binding Domains ◽  
Ccaat Box

CBTF122is a subunit of theXenopusCCAAT box transcription factor complex and a member of a family of double-stranded RNA-binding proteins that function in both transcriptional and post-transcriptional control. Here we identify a region of CBTF122containing the double-stranded RNA-binding domains that is capable of binding either RNA or DNA. We show that these domains bind A-form DNA in preference to B-form DNA and that the -59 to -31 region of the GATA-2 promoter (anin vivotarget of CCAAT box transcription factor) adopts a partial A-form structure. Mutations in the RNA-binding domains that inhibit RNA binding also affect DNA bindingin vitro. In addition, these mutations alter the ability of CBTF122fusions with engrailed transcription repressor and VP16 transcription activator domains to regulate transcription of the GATA-2 genein vivo. These data support the hypothesis that the double-stranded RNA-binding domains of this family of proteins are important for their DNA binding bothin vitroandin vivo.

scholarly journals Cooperativity boosts affinity and specificity of proteins with multiple RNA-binding domains

2021 ◽  
Author(s):  
Simon H. Stitzinger ◽  
Salma Sohrabi-Jahromi ◽  
Johannes Söding
Keyword(s):  
High Throughput ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Motif ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Rna Binding Domains

AbstractNumerous cellular processes rely on the binding of proteins with high affinity to specific sets of RNAs. Yet most RNA binding domains display low specificity and affinity, to the extent that for most RNA-binding domains, the enrichment of the best binding motif measured by high-throughput RNA SELEX or RNA bind-n-seq is usually below 10-fold, dramatically lower than that of DNA-binding domains. Here, we develop a thermodynamic model to predict the binding affinity for proteins with any number of RNA-binding domains given the affinities of their isolated domains. For the four proteins in which affinities for individual domains have been measured the model predictions are in good agreement with experimental values. The model gives insight into how proteins with multiple RNA-binding domains can reach affinities and specificities orders of magnitude higher than their individual domains. Our results contribute towards resolving the conundrum of missing specificity and affinity of RNA binding proteins and underscore the need for bioinformatic methods that can learn models for multi-domain RNA binding proteins from high-throughput in-vitro and in-vivo experiments.

In Caenorhabditis elegans, the RNA-Binding Domains of the Cytoplasmic Polyadenylation Element Binding Protein FOG-1 Are Needed to Regulate Germ Cell Fates

Genetics ◽  
2001 ◽  
Vol 159 (4) ◽  
pp. 1617-1630
Author(s):  
Suk-Won Jin ◽  
Nancy Arno ◽  
Adam Cohen ◽  
Amy Shah ◽  
Qijin Xu ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Germ Cell ◽  
Rna Binding ◽  
Dominant Negative ◽  
Missense Mutations ◽  
Biochemical Tests ◽  
Cell Fates ◽  
Cytoplasmic Polyadenylation ◽  
Binding Domains ◽  
Rna Binding Domains

Abstract FOG-1 controls germ cell fates in the nematode Caenorhabditis elegans. Sequence analyses revealed that FOG-1 is a cytoplasmic polyadenylation element binding (CPEB) protein; similar proteins from other species have been shown to bind messenger RNAs and regulate their translation. Our analyses of fog-1 mutations indicate that each of the three RNA-binding domains of FOG-1 is essential for activity. In addition, biochemical tests show that FOG-1 is capable of binding RNA sequences in the 3′-untranslated region of its own message. Finally, genetic assays reveal that fog-1 functions zygotically, that the small fog-1 transcript has no detectable function, and that missense mutations in fog-1 cause a dominant negative phenotype. This last observation suggests that FOG-1 acts in a complex, or as a multimer, to regulate translation. On the basis of these data, we propose that FOG-1 binds RNA to regulate germ cell fates and that it does so by controlling the translation of its targets. One of these targets might be the fog-1 transcript itself.

The N-terminal domain of Sxl protein disrupts Sxl autoregulation in females and promotes female-specific splicing of tra in males

Development ◽  
1999 ◽  
Vol 126 (13) ◽  
pp. 2841-2853 ◽  
Author(s):  
G. Deshpande ◽  
G. Calhoun ◽  
P.D. Schedl
Keyword(s):  
Fusion Protein ◽  
Rna Binding ◽  
Chimeric Protein ◽  
Dominant Negative ◽  
Terminal Domain ◽  
Binding Domains ◽  
Transcriptional Regulatory ◽  
Dominant Negative Activity ◽  
Female Specific

Sex determination in Drosophila depends upon the post-transcriptional regulatory activities of the Sex-lethal (Sxl) gene. Sxl maintains the female determined state and activates female differentiation pathways by directing the female-specific splicing of Sxl and tra pre-mRNAs. While there is compelling evidence that Sxl proteins regulate splicing by directly binding to target RNAs, previous studies indicate that the two Sxl RNA-binding domains are not in themselves sufficient for biological activity and that an intact N-terminal domain is also critical for splicing function. To further investigate the functions of the Sxl N terminus, we ectopically expressed a chimeric protein consisting of the N-terminal 99 amino acids fused to ss-galactosidase. The Nss-gal fusion protein behaves like a dominant negative, interfering with the Sxl autoregulatory feedback loop and killing females. This dominant negative activity can be attributed to the recruitment of the fusion protein into the large Sxl:Snf splicing complexes that are found in vivo and the consequent disruption of these complexes. In addition to the dominant negative activity, the Nss-gal fusion protein has a novel gain-of-function activity in males: it promotes the female-specific processing of tra pre-mRNAs. This novel activity is discussed in light of the blockage model for the tra splicing regulation.

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