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ABSTRACT: The FET family of proteins is composed of FUS/TLS, EWS/EWSR1, and TAF15 and possesses RNA- and DNA-binding capacities. The FET-proteins are involved in transcriptional regulation and RNA processing, and FET-gene deregulation is associated with development of cancer and protein granule formations in amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and trinucleotide repeat expansion diseases. We here describe a comparative characterization of FET-protein localization and gene regulatory functions. We show that FUS and TAF15 locate to cellular stress granules to a larger extend than EWS. FET-proteins have no major importance for stress granule formation and cellular stress responses, indicating that FET-protein stress granule association most likely is a downstream response to cellular stress. Gene expression analyses showed that the cellular response towards FUS and TAF15 reduction is relatively similar whereas EWS reduction resulted in a more unique response. The presented data support that FUS and TAF15 are more functionally related to each other, and that the FET-proteins have distinct functions in cellular signaling pathways which could have implications for the neurological disease pathogenesis.
PLoS ONE 01/2012; 7(9):e46251. · 4.09 Impact Factor
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ABSTRACT: The response of cells to changes in their environment often requires coregulation of gene networks, but little is known about how this can occur at the post-transcriptional level. An important example of post-transcriptional coregulation is the selective translational regulation in response to growth conditions of mammalian mRNAs that encode protein biosynthesis factors and contain hallmark 5'-terminal oligopyrimidine tracts (5'TOP). However, the responsible trans-factors and the mechanism by which they coregulate 5'TOP mRNAs have remained elusive. Here we identify stress granule-associated TIA-1 and TIAR proteins as key factors in human 5'TOP mRNA regulation, which upon amino acid starvation assemble onto the 5' end of 5'TOP mRNAs and arrest translation at the initiation step, as evidenced by TIA-1/TIAR-dependent 5'TOP mRNA translation repression, polysome release, and accumulation in stress granules. This requires starvation-mediated activation of the GCN2 (general control nonderepressible 2) kinase and inactivation of the mTOR (mammalian target of rapamycin) signaling pathway. Our findings provide a mechanistic explanation to the long-standing question of how the network of 5'TOP mRNAs are coregulated according to amino acid availability, thereby allowing redirection of limited resources to mount a nutrient deprivation response. This presents a fundamental example of how a group of mRNAs can be translationally coregulated in response to changes in the cellular environment.
Genes & development 10/2011; 25(19):2057-68. · 12.08 Impact Factor
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ABSTRACT: Transcription and pre-mRNA splicing are interdependent events. Although mechanisms governing the effects of transcription on splicing are becoming increasingly clear, the means by which splicing affects transcription remain elusive. Using cell lines stably expressing HIV-1 or beta-globin mRNAs, harboring wild-type or various 5' splice site mutations, we demonstrate a strong positive correlation between splicing efficiency and transcription activity. Interestingly, a 5' splice site can stimulate transcription even in the absence of splicing. Chromatin immunoprecipitation experiments show enhanced promoter docking of transcription initiation factors TFIID, TFIIB, and TFIIH on a gene containing a functional 5' splice site. In addition to their promoter association, the TFIID and TFIIH components, TBP and p89, are specifically recruited to the 5' splice site region. Our data suggest a model in which a promoter-proximal 5' splice site via its U1 snRNA interaction can feed back to stimulate transcription initiation by enhancing pre-initiation complex assembly.
Molecular Cell 03/2008; 29(2):271-8. · 14.18 Impact Factor
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ABSTRACT: A complex mRNA splicing pattern, which remains to be fully characterized, influences HIV-1 gene expression. In this study, poor envelope expression of a primary HIV-1 isolate was observed and linked to increased splicing of the two coding exons of tat/rev. The substitution of a nucleotide G, located 28 nucleotides upstream of the splice acceptor site SA7 in the recently identified intron splicing silencer sequence, was found to be responsible for the poor envelope expression. A single nucleotide substitution of G with A at this position results in a poor envelope expression phenotype. Moreover, substitution of the nucleotide G with any other nucleotide in an infectious HIV-1 proviral clone, HXB2RU3, results in poor envelope expression. The substitution of this nucleotide reduces the hnRNP A1 binding affinity but increases the splicing of env mRNA. The nucleotide G at this position is highly conserved among HIV-1 isolates and appears to play a critical role in HIV-1 splicing.
AIDS Research and Human Retroviruses 02/2006; 22(1):76-82. · 2.25 Impact Factor
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ABSTRACT: The untranslated leader of the dimeric HIV-1 RNA genome is folded into a complex structure that plays multiple and essential roles in the viral replication cycle. Here, we have investigated secondary and tertiary structural elements within the 5' 744 nucleotides of the HIV-1 genome using a combination of bioinformatics, enzymatic probing, native gel electrophoresis, and UV-crosslinking experiments. We used a recently developed RNA folding algorithm (Pfold) to predict the common secondary structure of an alignment of 20 divergent HIV-1 sequences. Combining this analysis with biochemical data, we present a secondary structure model for the entire 744 nucleotide fragment, which incorporates previously recognized and novel structural elements. In particular, our data provided strong evidence for a long-distance interaction between the region encompassing the AUG Gag initiation codon and an upstream region and we demonstrate that this feature is highly conserved in distantly related human and animal retroviruses. To obtain information about tertiary interactions we applied an intramolecular UV-crosslinking strategy and identified a novel tertiary interaction within the PBS hairpin structure.
Journal of Molecular Biology 03/2004; 336(2):369-79. · 4.00 Impact Factor
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ABSTRACT: The human immunodeficiency virus type 1 (HIV-1) particle contains two identical RNA strands, each corresponding to the entire genome. The 5' untranslated region (UTR) of each RNA strand contains extensive secondary and tertiary structures that are instrumental in different steps of the viral replication cycle. We have characterized the 5' UTRs of nine different HIV-1 isolates representing subtypes A through G and, by comparing their homodimerization and heterodimerization potentials, found that complementarity between the palindromic sequences in the dimerization initiation site (DIS) hairpins is necessary and sufficient for in vitro dimerization of two subtype RNAs. The 5' UTR sequences were used to design donor and acceptor templates for a coupled in vitro dimerization-reverse transcription assay. We showed that template switching during reverse transcription is increased with a matching DIS palindrome and further stimulated proportional to the level of homology between the templates. The presence of the HIV-1 nucleocapsid protein NCp7 increased the template-switching efficiency for matching DIS palindromes twofold, whereas the recombination efficiency was increased sevenfold with a nonmatching palindrome. Since NCp7 did not effect the dimerization of nonmatching palindromes, we concluded that the protein most likely stimulates the strand transfer reaction. An analysis of the distribution of template-switching events revealed that it occurs throughout the 5' UTR. Together, these results demonstrate that the template switching of HIV-1 reverse transcriptase occurs frequently in vitro and that this process is facilitated mainly by template proximity and the level of homology.
Journal of Virology 04/2003; 77(5):3020-30. · 5.40 Impact Factor
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ABSTRACT: The removal of the second intron in the HIV-1 rev/tat pre-mRNAs, which involves the joining of splice site SD4 to SA7, is inhibited by hnRNP A1 by a mechanism that requires the intronic splicing silencer (ISS) and the exon splicing silencer (ESS3). In this study, we have determined the RNA secondary structure and the hnRNP A1 binding sites within the 3' splice site region by phylogenetic comparison and chemical/enzymatic probing. A biochemical characterization of the RNA/protein complexes demonstrates that hnRNP A1 binds specifically to primarily three sites, the ISS, a novel UAG motif in the exon splicing enhancer (ESE) and the ESS3 element, which are all situated in experimentally supported stem loop structures. A mutational analysis of the ISS region revealed that the core hnRNP A1 binding site directly overlaps with a major branchpoint used in splicing to SA7, thereby providing a direct explanation for the inhibition of U2 snRNP association with the pre-mRNA by hnRNP A1. Binding of hnRNP A1 to the ISS core site is inhibited by RNA structure but strongly stimulated by the exonic silencer, ESS3. Moreover, the ISS also stimulate binding of hnRNP A1 to the exonic splicing regulators ESS3 and the ESE. Our results suggest a model where a network is formed between hnRNP A1 molecules situated at discrete sites in the intron and exon and that these interactions preclude the recognition of essential splicing signals including the branch point.
RNA 12/2002; 8(11):1401-15. · 5.09 Impact Factor
