nteractions of INS (CRS) Elements and the Splicing Machinery Regulate the Production of Rev-responsive mRNAs

Mikaélian, Ivan; Krieg, Marion; Gait, Michael J.; Karn, Jonathan

doi:10.1006/jmbi.1996.0160

Cited by 60 publications

(45 citation statements)

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“…This sequence acts synergistically with the Rev response element to promote Rev-dependent export of unspliced transcripts ( Fig. 2A) (46). Interestingly, the INS sequence has been shown to be a specific binding site for both hnRNP A1 and for an unknown protein of 50 kDa (34).…”

Section: Resultsmentioning

confidence: 98%

Determination of the RNA Binding Specificity of the Heterogeneous Nuclear Ribonucleoprotein (hnRNP) H/H′/F/2H9 Family

Caputi

Zahler

2001

Journal of Biological Chemistry

188

176

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Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) H protein family, H, H, F, and 2H9, are involved in pre-mRNA processing. We analyzed the assembly of these proteins from splicing extracts onto four RNA regulatory elements as follows: a high affinity hnRNP A1-binding site (WA1), a sequence involved in Rev-dependent export (p17gag INS), an exonic splicing silencer from the ␤-tropomyosin gene, and an intronic splicing regulator (downstream control sequence (DCS) from the c-src gene. The entire family binds the WA1, instability (INS), and ␤-tropomyosin substrates, and the core-binding site for each is a run of three G residues followed by an A. Transfer of small regions containing this sequence to a substrate lacking hnRNP H binding activity is sufficient to promote binding of all family members. The c-src DCS has been shown to assemble hnRNP H, not hnRNP F, from HeLa cell extracts, and we show that hnRNP 2H9 does not bind this element. The DCS contains five G residues followed by a C. Mutation of the C to an A changes the specificity of the DCS from a substrate that binds only hnRNP H/H to a binding site for all hnRNP H family members. We conclude that the sequence GGGA is recognized by all hnRNP H family proteins.Some proteins of the heterogeneous nuclear ribonucleoprotein (hnRNP) 1 class were initially described as components of a nucleosome-like structure that protects and organizes nascent RNA polymerase II transcripts (1-3). Our understanding of hnRNP functions has broadened from this purely structural vision to include multiple aspects of mRNA biogenesis such as transcription, splicing, capping, polyadenylation, nuclear transport, and mRNA stability. More than 30 hnRNPs have been identified so far. Many hnRNPs have a modular structure characterized by one or more RNA binding domains and by one or more auxiliary domains that are frequently enriched in a few amino acids, mainly glycines. The RNA recognition motif (RRM), the arginine-rich motif, the KH domain, and the RGG box are some of the main motifs that are found in hnRNP proteins (4).The precise roles of hnRNP proteins in gene expression are likely to be reflected by their RNA binding specificity. Therefore, it is important to have a clear understanding of the RNA binding specificities and affinities of the different family members. Early studies demonstrated that several hnRNPs have affinities for immobilized ribohomopolymers such as poly(G) bound by hnRNPs E, H, F, and M, poly(C) bound by hnRNPs K and J, and poly(U) bound by hnRNPs C and M (5). Subsequently, several hnRNPs were observed to bind specific RNA sequences by UV-mediated cross-linking. Furthermore, utilizing a selection and amplification approach from pools of random RNAs, high affinity binding sequences for hnRNPs A1 (6) and C (7), have been identified. The binding properties of hnRNP A1 are the best characterized so far. Several reports (1, 8 -12) have shown that hnRNP A1 and other members of the hnRNP A/B group (hnRNP A1b, A2, and B1) share common RNA substrate specificities...

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Section: Resultsmentioning

confidence: 98%

Determination of the RNA Binding Specificity of the Heterogeneous Nuclear Ribonucleoprotein (hnRNP) H/H′/F/2H9 Family

Caputi

Zahler

2001

Journal of Biological Chemistry

188

176

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“…Rev dependence may be related to the presence of constitutive repressive elements in the viral transcripts encoding structural genes (Brighty and Rosenberg, 1994;Churchill et al, 1996;Cochrane et al, 1991;Felber et al, 1989;Mikaelian et al, 1996;Nasioulas et al, 1994;Schwartz et al, 1992a,b). Such sequences have been reported to act independent of splicing signals (Nasioulas et al, 1994), to over lap in part with the RRE (Brighty and Rosenberg, 1994;Churchill et al, 1996), and to require cooperation with additional viral repressive sequences (Mikaelian et al, 1996). However, the function of 3' CRS may be unique to the requirements of HTLV-1.…”

Section: Discussionmentioning

confidence: 99%

Nucleocytoplasmic transport of HTLV-1 RNA is regulated by two independent LTR encoded nuclear retention elements

et al. 1998

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Appropriate expression of HTLV-1 genes requires transcriptional transactivation by Tax and post-transcriptional regulation by Rex, both mediated by LTR encoded RNA sequences. Using a combination of deletion mutagenesis, Rex-reporter CAT assays,¯uores-cence in situ hybridization (FISH) and confocal laser scanning microscopy it was established that in the absence of Rex, CAT mRNAs harboring HTLV-1 LTR sequences were unable to leave the nucleus. Deletion of the known U5 encoded cis-acting repressing sequence (CRS) led to a partial release of nuclear retention. A novel regulatory element overlapping the 3' Rex responsive element (RxRE) region was shown to prevent export and expression of these transcripts. Deletion of both the 5' LTR encoded CRS and 3' LTR encoded downstream repressive sequence (3' CRS) led to constitutive mRNA nuclear export and gene expression, independently of Rex. The locations of the two regulatory elements indicate that while the 5' CRS selectively acts to hinder export of unspliced transcripts, the 3' CRS has the capacity to induce nuclear retention of all HTLV-1 transcripts, and therefore could potentially contribute to viral latency in infected cells.

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“…Tatinducible gene expression can be achieved by using the HIV LTR promoter or heterochimeric promoters containing the HIV TAR element (197,198). Rev-inducible production of the therapeutic gene product may be achieved by designing, within the coding region of the therapeutic gene, instability elements (INS) and the RRE (199). INS sequences decrease mRNA stability, whereas the binding of HIV Rev to the RRE overrides this negative effect and increases the half-life of the transcripts produced, making the gene product Rev-inducible (199).…”

Section: Gene Delivery and Expressionmentioning

confidence: 99%

“…Rev-inducible production of the therapeutic gene product may be achieved by designing, within the coding region of the therapeutic gene, instability elements (INS) and the RRE (199). INS sequences decrease mRNA stability, whereas the binding of HIV Rev to the RRE overrides this negative effect and increases the half-life of the transcripts produced, making the gene product Rev-inducible (199). MLV-based retroviral vectors allowing Tat-and Rev-inducible gene expression have been constructed (200,201).…”

Section: Gene Delivery and Expressionmentioning

confidence: 99%

Potential nuclease-based strategies for HIV gene therapy

Singwi

Joshi²

2000

Front Biosci

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nteractions of INS (CRS) Elements and the Splicing Machinery Regulate the Production of Rev-responsive mRNAs

Cited by 60 publications

References 0 publications

Determination of the RNA Binding Specificity of the Heterogeneous Nuclear Ribonucleoprotein (hnRNP) H/H′/F/2H9 Family

Determination of the RNA Binding Specificity of the Heterogeneous Nuclear Ribonucleoprotein (hnRNP) H/H′/F/2H9 Family

Nucleocytoplasmic transport of HTLV-1 RNA is regulated by two independent LTR encoded nuclear retention elements

Potential nuclease-based strategies for HIV gene therapy

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