Distinct functions of the closely related tandem RNA-recognition motifs of hnRNP A1

Mayeda, Akila; Munroe, Stephen H.; Xu, Rui; Krainer, Adrian R.

doi:10.1017/s135583829898089x

Cited by 50 publications

(51 citation statements)

References 48 publications

(37 reference statements)

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“…The Gly Domain of Protein hnRNP A1 Increases the Affinity for SLS3-In agreement with previous results (23,39), our gelshift and footprinting experiments reveal a much stronger affinity of protein hnRNP A1 for SLS3, as compared with protein UP1. Hence, the Gly domain of protein hnRNP A1 participates in some way to the interaction with SLS3.…”

Section: Discussionsupporting

confidence: 92%

“…Hence, the Gly domain of protein hnRNP A1 participates in some way to the interaction with SLS3. The reinforced binding may be due both to the RNA binding capacity of the Gly domain (39) and its capacity to establish protein-protein interactions between hnRNP A1 molecules (19,40). Such additional interactions should be necessary to stabilize the association of protein hnRNP A1 with the 5Ј strand of loop II and the segment at the SLS2-SLS3 junction.…”

Section: Discussionmentioning

confidence: 99%

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Biochemical and NMR Study on the Competition between Proteins SC35, SRp40, and Heterogeneous Nuclear Ribonucleoprotein A1 at the HIV-1 Tat Exon 2 Splicing Site

Hallay¹,

Locker

Ayadi³

et al. 2006

Journal of Biological Chemistry

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The human immunodeficiency virus, type 1, Tat protein plays a key role in virus multiplication. Because of its apoptotic property, its production is highly controlled. It depends upon the A3 splicing site utilization. A key control of site A3 activity is the ESS2 splicing silencer, which is located within the long stem-loop structure 3 (SLS3), far downstream from site A3. Here, by enzymatic footprints, we demonstrate the presence of several heterogeneous nuclear ribonucleoprotein (hnRNP) A1-binding sites on SLS3 and show the importance of the C-terminal Gly domain of hnRNP A1 in the formation of stable complexes containing several hnRNP A1 molecules bound on SLS3. Mutations in each of the UAG triplets in ESS2 strongly reduce the overall hnRNP A1 binding, showing the central role of ESS2 in hnRNP A1 assembly on SLS2-SLS3. Using NMR spectroscopy, we demonstrate the direct interaction of ESS2 with the RNA recognition motifs domains of hnRNP A1. This interaction has limited effect on the RNA two-dimensional structure. The SR proteins SC35 and SRp40 were found previously to be strong activators of site A3 utilization. By enzymatic and chemical footprints, we delineate their respective binding sites on SLS2 and SLS3 and find a strong similarity between the hnRNP A1-, SC35-, and SRp40-binding sites. The strongest SC35-binding site only has a modest contribution to site A3 activation. Hence, the main role of SR proteins at site A3 is to counteract hnRNP A1 binding on ESS2 and ESE2. Indeed, we found that ESE2 has inhibitory properties because of its ability to bind hnRNP A1.Retrovirus RNAs are transcribed from an integrated proviral genome. Part of the transcripts has to be transported to the cytoplasm in an unspliced form and is used as mRNAs for the production of the viral, Gag, Gag-Pol, and Env protein precursors and as genomic RNAs for new virion assembly. Another part of the transcripts has to be spliced for production of the Tat, Rev, Nef, Vif, and Vpr proteins. Because of the presence of four splicing donor sites (5Ј ss) 4 and eight splicing acceptor sites (3Ј ss) in HIV-1 RNA, the splicing machinery of infected cells generates at least 40 distinct mRNAs from a unique RNA primary transcript (1). The relative abundance of these mRNAs depends greatly on the relative efficiencies of the 3Ј ss, which are suboptimal (2-11). During the early phase of cell infection, the five 3Ј ss (A3, A4c, A4a, A4b, and A5) located in a small central part of the viral RNA are used for production of the tat, rev, and nef mRNAs (1). All the tat mRNAs are spliced at site A3. The rev mRNAs are spliced at sites A4a, A4b, or A4c, and most of the nef mRNAs are spliced at site A5 (1, 12).The Tat protein plays a key role in virus multiplication as it is needed for production of full-length HIV-1 transcripts (13). However, because of the apoptotic activity of this protein on both the infected cells and the neighboring cells (13-15), virus HIV-1 strongly controls its production. In both lymphoid and nonlymphoid infected cells, the steady-state le...

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Biochemical and NMR Study on the Competition between Proteins SC35, SRp40, and Heterogeneous Nuclear Ribonucleoprotein A1 at the HIV-1 Tat Exon 2 Splicing Site

Hallay¹,

Locker

Ayadi³

et al. 2006

Journal of Biological Chemistry

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“…So are, for example, the tandem RRMs of hnRNPA1 not equivalent (Mayeda et al, 1998). Although both are required for alternative splicing function, each RRM plays distinct roles; RRM2 can function in alternative splicing when duplicated, whereas RRM1 cannot.…”

Section: Discussionmentioning

confidence: 99%

A key role for stress-induced satellite III transcripts in the relocalization of splicing factors into nuclear stress granules

Metz

Soret

Vourc’h

et al. 2004

Journal of Cell Science

116

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Exposure of cells to stressful conditions results in the rapid synthesis of a subset of specialized proteins termed heat shock proteins (HSPs) which function in protecting the cell against damage. The stress-induced activation of hsp genes is controlled by the heat shock transcription factor 1 (HSF1). At the cellular level, one of the most striking effects of stress is the rapid and reversible redistribution of HSF1 into a few nuclear structures termed nuclear stress granules which form primarily on the 9q12 locus in humans. Within these structures, HSF1 binds to satellite III repeated elements and drives the RNA polymerase II-dependent transcription of these sequences into stable RNAs which remain associated with the 9q12 locus for a certain time after synthesis. Other proteins, in particular splicing factors, were also shown to relocalize to the granules upon stress. Here, we investigated the role of stress-induced satellite III transcripts in the relocalization of splicing factors to the granules. We show that the recruitment of the two serine/arginine-rich (SR) proteins SF2/ASF and SRp30c requires the presence of stress-induced satellite III transcripts. In agreement with these findings, we identified the second RNA-recognition motif (RRM2) of hSF2/ASF as the motif required for the targeting to the granules, and we showed by immunoprecipitation that the endogenous hSF2/ASF protein is present in a complex with satellite III transcripts in stressed cells in vivo. Interestingly, satellite III transcripts also immunoprecipitate together with small nuclear ribonucleoproteins (snRNPs) in vivo whereas the intronless hsp70 transcripts do not, supporting the proposal that these transcripts are subject to splicing. Altogether, these data highlight the central role for satellite III transcripts in the targeting and/or retention of splicing factors into the granules upon stress.

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“…Whether the reduced activity of dFMR1 with an inactivated KH domain comes from a necessity for both KH domains to function together to bind RNA or whether each KH domain may have some individual functions is not clear. The tandem RNA-binding domains of hnRNP A1 have been shown to have both shared and distinct functions (39). Identification of in vivo substrates for dFMR1 will be needed to further address this issue.…”

Section: Discussionmentioning

confidence: 99%

Characterization of dFMR1, a Drosophila melanogaster Homolog of the Fragile X Mental Retardation Protein

Wan

Dockendorff

Jongens

et al. 2000

Molecular and Cellular Biology

260

261

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Fragile X syndrome is the most common inherited form of mental retardation. It is caused by loss of FMR1 gene activity due to either lack of expression or expression of a mutant form of the protein. In mammals, FMR1 is a member of a small protein family that consists of FMR1, FXR1, and FXR2. All three members bind RNA and contain sequence motifs that are commonly found in RNA-binding proteins, including two KH domains and an RGG box. The FMR1/FXR proteins also contain a 60S ribosomal subunit interaction domain and a protein-protein interaction domain which mediates homomer and heteromer formation with each family member. Nevertheless, the specific molecular functions of FMR1/FXR proteins are unknown. Here we report the cloning and characterization of a Drosophila melanogaster homolog of the mammalian FMR1/FXR gene family. This first invertebrate homolog, termed dfmr1, has a high degree of amino acid sequence identity/ similarity with the defined functional domains of the FMR1/FXR proteins. The dfmr1 product binds RNA and is similar in subcellular localization and embryonic expression pattern to the mammalian FMR1/FXR proteins. Overexpression of dfmr1 driven by the UAS-GAL4 system leads to apoptotic cell loss in all adult Drosophila tissues examined. This phenotype is dependent on the activity of the KH domains. The ability to induce a dominant phenotype by overexpressing dfmr1 opens the possibility of using genetic approaches in Drosophila to identify the pathways in which the FMR1/FXR proteins function.Fragile X syndrome is the most common form of hereditary mental retardation whose effects are traced to the loss of function of a single gene, named FMR1. This syndrome affects approximately 1 in 5,000 male births and is globally distributed throughout the human population (19,42,61). In most cases, the disease results from the repression of FMR1 gene expression that is due to an expansion of a CGG trinucleotide repeat in the 5Ј untranslated region of the gene (25,28,36,45,65,66,73). Subsequent methylation of this expanded repeat results in transcriptional silencing of the FMR1 gene (7, 43). A few fragile X patients with partial or complete deletions of the FMR1 gene have been identified, and these patients have phenotypes similar to those affected by the trinucleotide repeat expansion (26, 68). One patient who has a single point mutation in the FMR1 gene that replaces an isoleucine residue at amino acid 304 with asparagine (I304N) exhibits a particularly severe fragile X phenotype (17). The severity of the phenotype observed in this patient has prompted the suggestion that the I304N substitution results in a dominant-negative form of FMR1 protein (22, 53).The FMR1 protein binds RNA in vitro and contains two types of RNA-binding motifs, KH domains and an RGG box (10, 35, 55). The RNA-binding activity of FMR1 appears to be selective. It has been estimated that FMR1 interacts with about 4% of human fetal brain mRNAs, including its own mRNA (4). The importance of the RNA-binding activity to the function of the FMR1 ...

show abstract

Distinct functions of the closely related tandem RNA-recognition motifs of hnRNP A1

Cited by 50 publications

References 48 publications

Biochemical and NMR Study on the Competition between Proteins SC35, SRp40, and Heterogeneous Nuclear Ribonucleoprotein A1 at the HIV-1 Tat Exon 2 Splicing Site

Biochemical and NMR Study on the Competition between Proteins SC35, SRp40, and Heterogeneous Nuclear Ribonucleoprotein A1 at the HIV-1 Tat Exon 2 Splicing Site

A key role for stress-induced satellite III transcripts in the relocalization of splicing factors into nuclear stress granules

Characterization of dFMR1, a Drosophila melanogaster Homolog of the Fragile X Mental Retardation Protein

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