RNA helicase A (RHA) is a highly conserved DEAD-box protein that activates transcription, modulates RNA splicing and binds the nuclear pore complex. The life cycle of typical mRNA involves RNA processing and translation after ribosome scanning of a relatively unstructured 5' untranslated region (UTR). The precursor RNAs of retroviruses and selected cellular genes harbor a complex 5' UTR and use a yet-to-be-identified host post-transcriptional effector to stimulate efficient translation. Here we show that RHA recognizes a structured 5'-terminal post-transcriptional control element (PCE) of a retrovirus and the JUND growth-control gene. RHA interacts with PCE RNA in the nucleus and cytoplasm, facilitates polyribosome association and is necessary for its efficient translation. Our results reveal a previously unidentified role for RHA in translation and implicate RHA as an integrative effector in the continuum of gene expression from transcription to translation.
"We tested the contribution of simple de-repression by replacing the deletion mutant with the co-transfection of RNA helicase A (RHA). This DExH helicase (also called DHx9) has been reported to selectively stimulate translational expression from transcripts carrying structured 5′UTRs . Additionally, since sElk1 was reported in the rat PC12 cell line that expresses a 5′UTRL carrying a distinct exonII we also tested this variant in the assay (Figure 4C). "
[Show abstract][Hide abstract] ABSTRACT: Elk1 belongs to the ternary complex (TCF) subfamily of the ETS-domain transcription factors. Several studies have implicated an important function for Elk1 in the CNS including synaptic plasticity and cell differentiation. Whilst studying ELK1 gene expression in rat brain a 54 aa N-terminally truncated isoform lacking the DBD was observed on immunoblots. A similar protein was also detected in NGF differentiated PC12 cells. It was proposed that this protein, referred to as sElk1, arose due to a de-novo initiation event at the second AUG codon on the Elk1 ORF. Transient over-expression of sElk1 potentiated neurite growth in the PC12 model and induced differentiation in the absence of NGF, leading to the proposition that it may have a specific function in the CNS. Here we report on the translational expression from the mouse and rat transcript and compare it with our earlier published work on human. Results demonstrate that the previously observed sElk1 protein is a non-specific band arising from the antibody employed. The tight conservation of the internal AUG reported to drive sElk1 expression is in fact coupled to Elk1 protein function, a result consistent with the Elk1-SRE crystal structure. It is also supported by the observed conservation of this methionine in the DBD of all ETS transcription factors independent of the N- or C-terminal positioning of this domain. Reporter assays demonstrate that elements both within the 5'UTR and downstream of the AUGElk1 serve to limit 40S access to the AUGsElk1 codon.
PLoS ONE 07/2014; 9(7):e102890. DOI:10.1371/journal.pone.0102890 · 3.23 Impact Factor
"The significance of the repetitive unwinding must be considered in light of the diverse functions of RHA in the cell. RHA participates in the activation of transcription (19,20,33,35), mRNA splicing (18,36,37), and stimulation of translation (11). RHA has been found associated with the Rev/Rev-response element (RRE) complex (38), the nuclear pore complex (39) and the RNA-induced silencing complex (RISC) involved in the RNAi pathway (12). "
[Show abstract][Hide abstract] ABSTRACT: Helicases contribute to diverse biological processes including replication, transcription and translation. Recent reports
suggest that unwinding of some helicases display repetitive activity, yet the functional role of the repetitiveness requires
further investigation. Using single-molecule fluorescence assays, we elucidated a unique unwinding mechanism of RNA helicase
A (RHA) that entails discrete substeps consisting of binding, activation, unwinding, stalling and reactivation stages. This multi-step process is repeated many times by a single RHA molecule without dissociation, resulting in repetitive
unwinding/rewinding cycles. Our kinetic and mutational analysis indicates that the two double stand RNA binding domains at
the N-terminus of RHA are responsible for such repetitive unwinding behavior in addition to providing an increased binding
affinity to RNA. Further, the repetitive unwinding induces an efficient annealing of a complementary RNA by making the unwound
strand more accessible. The complex and unusual mechanism displayed by RHA may help in explaining how the repetitive unwinding
of helicases contributes to their biological functions.
Nucleic Acids Research 06/2014; 42(13). DOI:10.1093/nar/gku523 · 9.11 Impact Factor
"RNA helicase A, also denominated DHX9, is another RNA-binding protein with ATPase and RNA helicase activities. It also participates mRNA metabolism by activating several transcriptional pathways  and promoting translation of selected messenger RNA containing defined elements in the 5′UTR , . "
[Show abstract][Hide abstract] ABSTRACT: Most genes have multiple polyadenylation sites (PAS), which are often selected in a tissue-specific manner, altering protein products and affecting mRNA stability, subcellular localization and/or translability. Here we studied the polyadenylation mechanisms associated to the beta-adducin gene (Add2). We have previously shown that the Add2 gene has a very tight regulation of alternative polyadenylation, using proximal PAS in erythroid tissues, and a distal one in brain. Using chimeric minigenes and cell transfections we identified the core elements responsible for polyadenylation at the distal PAS. Deletion of either the hexanucleotide motif (Hm) or the downstream element (DSE) resulted in reduction of mature mRNA levels and activation of cryptic PAS, suggesting an important role for the DSE in polyadenylation of the distal Add2 PAS. Point mutation of the UG repeats present in the DSE, located immediately after the cleavage site, resulted in a reduction of processed mRNA and in the activation of the same cryptic site. RNA-EMSA showed that this region is active in forming RNA-protein complexes. Competition experiments showed that RNA lacking the DSE was not able to compete the RNA-protein complexes, supporting the hypothesis of an essential important role for the DSE. Next, using a RNA-pull down approach we identified some of the proteins bound to the DSE. Among these proteins we found PTB, TDP-43, FBP1 and FBP2, nucleolin, RNA helicase A and vigilin. All these proteins have a role in RNA metabolism, but only PTB has a reported function in polyadenylation. Additional experiments are needed to determine the precise functional role of these proteins in Add2 polyadenylation.
PLoS ONE 03/2013; 8(3):e58879. DOI:10.1371/journal.pone.0058879 · 3.23 Impact Factor
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