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

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.

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Heterogeneous nuclear ribonucleoprotein D0 contains transactivator and DNA-binding domains

Biochemical Journal ◽

10.1042/bj3480151 ◽

2000 ◽

Vol 348 (1) ◽

pp. 151-158 ◽

Cited By ~ 13

Author(s):

Mate TOLNAY ◽

Lajos BARANYI ◽

George C. TSOKOS

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Dna Binding ◽

Rna Binding ◽

Dna Binding Domains ◽

Heterogeneous Nuclear Ribonucleoprotein ◽

Binding Domains ◽

Double Stranded Dna ◽

Nuclear Ribonucleoprotein

Heterogeneous nuclear ribonucleoprotein D0 (hnRNP D0) is an abundant, ubiquitous protein that binds RNA and DNA sequences specifically, and has been implicated in the transcriptional regulation of the human complement receptor 2 gene. We found that in vivo expression of hnRNP D0-GAL4 fusion proteins increased the transcriptional activity of a GAL4-driven reporter gene, providing direct proof that hnRNP D0 possesses a transactivator domain. We found, using truncated hnRNP D0 proteins fused to GAL4, that 29 amino acids in the N-terminal region are critical for transactivation. We established, using a series of recombinant truncated hnRNP D0 proteins, that the tandem RNA-binding domains alone were not able to bind double-stranded DNA. Nevertheless, 24 additional amino acids of the C-terminus imparted sequence-specific DNA binding. Experiments using peptide-specific antisera supported the importance of the 24-amino-acid region in DNA binding, and suggested the involvement of the 19-amino-acid alternative insert which is present in isoforms B and D. The N-terminus had an inhibitory effect on binding of hnRNP D0 to single-stranded, but not to double-stranded, DNA. Although both recombinant hnRNP D0B and D0D bound DNA, only the B isoform recognized DNA in vivo. We propose that the B isoform of hnRNP D0 functions in the nucleus as a DNA-binding transactivator and has distinct transactivator and DNA-binding domains.

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NH2 terminus of PTB-associated splicing factor binds to the porcine P450scc IGF-I response element

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00057.2002 ◽

2002 ◽

Vol 283 (3) ◽

pp. E423-E427 ◽

Cited By ~ 16

Author(s):

Randall J. Urban ◽

Yvonne H. Bodenburg ◽

Thomas G. Wood

Keyword(s):

Amino Acid ◽

Transcriptional Activity ◽

Rna Binding ◽

Terminal Region ◽

Splicing Factor ◽

Amino Acid Residues ◽

Response Element ◽

Binding Domains ◽

Igf I ◽

Rna Binding Domains

An insulin-like growth factor (IGF) I response element (IGFRE) in the porcine P-450 cholesterol side-chain cleavage gene (P450scc) regulates transcription through the binding of two proteins, Sp1 and polypyrimidine tract-binding protein-associated splicing factor (PSF). PSF is a component of spliceosomes and contains RNA-binding domains. In this study, we localized the NH2-terminal amino acid residues necessary for binding of PSF to the IGFRE. Three COOH-terminal truncated proteins (aa 304, 214, and 134) of PSF were designed to empirically partition the NH2-terminal region while excluding the RNA-binding domains. Southwestern analysis showed that only the largest expressed truncated protein, P3, strongly bound the porcine P450scc IGFRE. Truncated PSF protein expression in Y1 adrenal cells showed that P3 repressed transcriptional activity of the IGFRE similar to full-length PSF, whereas P2 (minimal binding to the IGFRE) had no effect. In conclusion, the NH2-terminal region of PSF contains the amino acid residues necessary for binding to the porcine P450scc IGFRE and repressing the transcriptional activity of the element.

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Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

Molecular and Cellular Biology ◽

10.1128/mcb.7.1.294 ◽

1987 ◽

Vol 7 (1) ◽

pp. 294-304 ◽

Cited By ~ 30

Author(s):

D Pilgrim ◽

E T Young

Keyword(s):

Amino Acids ◽

Enzyme Activity ◽

Amino Acid ◽

Proteolytic Processing ◽

Mitochondrial Matrix ◽

Wild Type ◽

Mature Form ◽

Amino Terminal ◽

The Matrix

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

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The multiple RNA-binding domains of the mRNA poly(A)-binding protein have different RNA-binding activities.

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3419 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3419-3424 ◽

Cited By ~ 130

Author(s):

C G Burd ◽

E L Matunis ◽

G Dreyfuss

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Consensus Sequence ◽

Binding Activity ◽

Yeast Cells ◽

Yeast Saccharomyces Cerevisiae ◽

Amino Terminal ◽

Binding Domains ◽

Rna Binding Domains

The poly(A)-binding protein (PABP) is the major mRNA-binding protein in eukaryotes, and it is essential for viability of the yeast Saccharomyces cerevisiae. The amino acid sequence of the protein indicates that it consists of four ribonucleoprotein consensus sequence-containing RNA-binding domains (RBDs I, II, III, and IV) and a proline-rich auxiliary domain at the carboxyl terminus. We produced different parts of the S. cerevisiae PABP and studied their binding to poly(A) and other ribohomopolymers in vitro. We found that none of the individual RBDs of the protein bind poly(A) specifically or efficiently. Contiguous two-domain combinations were required for efficient RNA binding, and each pairwise combination (I/II, II/III, and III/IV) had a distinct RNA-binding activity. Specific poly(A)-binding activity was found only in the two amino-terminal RBDs (I/II) which, interestingly, are dispensable for viability of yeast cells, whereas the activity that is sufficient to rescue lethality of a PABP-deleted strain is in the carboxyl-terminal RBDs (III/IV). We conclude that the PABP is a multifunctional RNA-binding protein that has at least two distinct and separable activities: RBDs I/II, which most likely function in binding the PABP to mRNA through the poly(A) tail, and RBDs III/IV, which may function through binding either to a different part of the same mRNA molecule or to other RNA(s).

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

RNA ◽

10.1017/s135583829898102x ◽

1998 ◽

Vol 4 (12) ◽

pp. 1585-1598 ◽

Cited By ~ 19

Author(s):

KAI ZU ◽

MARTHA L. SIKES ◽

ANN L. BEYER

Keyword(s):

Rna Binding ◽

Binding Domains ◽

Rna Binding Domains

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

10.1101/466763 ◽

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.200301034 ◽

2003 ◽

Vol 161 (2) ◽

pp. 309-319 ◽

Cited By ~ 18

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.

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The multiple RNA-binding domains of the mRNA poly(A)-binding protein have different RNA-binding activities

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3419-3424.1991 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3419-3424

Author(s):

C G Burd ◽

E L Matunis ◽

G Dreyfuss

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Consensus Sequence ◽

Binding Activity ◽

Yeast Cells ◽

Yeast Saccharomyces Cerevisiae ◽

Amino Terminal ◽

Binding Domains ◽

Rna Binding Domains

The poly(A)-binding protein (PABP) is the major mRNA-binding protein in eukaryotes, and it is essential for viability of the yeast Saccharomyces cerevisiae. The amino acid sequence of the protein indicates that it consists of four ribonucleoprotein consensus sequence-containing RNA-binding domains (RBDs I, II, III, and IV) and a proline-rich auxiliary domain at the carboxyl terminus. We produced different parts of the S. cerevisiae PABP and studied their binding to poly(A) and other ribohomopolymers in vitro. We found that none of the individual RBDs of the protein bind poly(A) specifically or efficiently. Contiguous two-domain combinations were required for efficient RNA binding, and each pairwise combination (I/II, II/III, and III/IV) had a distinct RNA-binding activity. Specific poly(A)-binding activity was found only in the two amino-terminal RBDs (I/II) which, interestingly, are dispensable for viability of yeast cells, whereas the activity that is sufficient to rescue lethality of a PABP-deleted strain is in the carboxyl-terminal RBDs (III/IV). We conclude that the PABP is a multifunctional RNA-binding protein that has at least two distinct and separable activities: RBDs I/II, which most likely function in binding the PABP to mRNA through the poly(A) tail, and RBDs III/IV, which may function through binding either to a different part of the same mRNA molecule or to other RNA(s).

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

Journal of Biological Chemistry ◽

10.1074/jbc.m406107200 ◽

2004 ◽

Vol 279 (50) ◽

pp. 52447-52455 ◽

Cited By ~ 12

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.

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