A novel role of RNA helicase A in regulation of translation of type I collagen mRNAs

Type I collagen is composed of two α1(I) polypeptides and one α2(I) polypeptide and is the most abundant protein in the human body. Expression of type I collagen is primarily controlled at the level of mRNA stability and translation. Coordinated translation of α(I) and α2(I) mRNAs is necessary for efficient folding of the corresponding peptides into the collagen heterotrimer. In the 5' untranslated region (5' UTR), collagen mRNAs have a unique 5' stem-loop structure (5' SL). La ribonucleoprotein domain family member 6 (LARP6) is the protein that binds 5' SL with high affinity and specificity and coordinates their translation. Here we show that RNA helicase A (RHA) is tethered to the 5' SL of collagen mRNAs by interaction with the C-terminal domain of LARP6. In vivo, collagen mRNAs immunoprecipitate with RHA in an LARP6-dependent manner. Knockdown of RHA prevents formation of polysomes on collagen mRNAs and dramatically reduces synthesis of collagen protein, without affecting the level of the mRNAs. A reporter mRNA with collagen 5' SL is translated three times more efficiently in the presence of RHA than the same reporter without the 5' SL, indicating that the 5' SL is the cis-acting element conferring the regulation. During activation of quiescent cells into collagen-producing cells, expression of RHA is highly up-regulated. We postulate that RHA is recruited to the 5' UTR of collagen mRNAs by LARP6 to facilitate their translation. Thus, RHA has been discovered as a critical factor for synthesis of the most abundant protein in the human body.     

The adaptor Grb7 links netrin-1 signaling to regulation of mRNA translation

We previously reported a novel biological activity of Netrin-1 in translational stimulation of kappa opioid receptor (KOR). We now identify Grb7 as a new RNA-binding protein that serves as the molecular adaptor for transmitting Netrin-1 signals, through focal adhesion kinase (FAK), to the translation machinery. Grb7 binds specifically to the first stem loop of kor mRNA 5'-UTR through a new RNA-binding domain located in its amino terminus. Upon binding to its capped, target mRNA, Grb7 blocks the recruitment of eIF4E, rendering mRNA untranslatable. The RNA-binding and translation-repressive activity is reduced by FAK-mediated hyperphosphorylation on two tyrosine residues of its carboxyl terminus. This study reports an adaptor protein Grb7 that transmits the stimulating signals of Netrin-1 to the translational machinery to rapidly regulate mRNA translation.     

Solution structures of the double-stranded RNA-binding domains from RNA helicase A

RNA helicase A (RHA) is a highly conserved protein with multifaceted functions in the gene expression of cellular and viral mRNAs. RHA recognizes highly structured nucleotides and catalytically rearranges the various interactions between RNA, DNA, and protein molecules to provide a platform for the ribonucleoprotein complex. We present the first solution structures of the double-stranded RNA-binding domains (dsRBDs), dsRBD1 and dsRBD2, from mouse RHA. We discuss the binding mode of the dsRBDs of RHA, in comparison with the known dsRBD structures in their complexes. Our structural data provide important information for the elucidation of the molecular reassembly mediated by RHA.     

Phosphorylation of p68 RNA helicase regulates RNA binding by the C-terminal domain of the protein

We previously reported ATPase, RNA unwinding, and RNA-binding activities of recombinant p68 RNA helicase that was expressed in Escherichia coli. Huang et al. The recombinant protein bound both single-stranded (ss) and double-stranded (ds) RNAs. To further characterize the substrate RNA binding by p68 RNA helicase, we expressed and purified the recombinant N-terminal and C-terminal domains of the protein. RNA-binding property and protein phosphorylation of the recombinant domains of p68 were analyzed. Our data demonstrated that the C-terminal domain of p68 RNA helicase bound ssRNA. More interestingly, the C-terminal domain was a target of protein kinase C (PKC). Phosphorylation of the C-terminal domain of p68 abolished its RNA binding. Based on our observations, we propose that the C-terminal domain is an RNA substrate binding site for p68. The protein phosphorylation by PKC regulates the RNA binding of p68 RNA helicase, which consequently controls the enzymatic activities of the protein.     

RNA-binding activity of translation initiation factor eIF4G1 from Saccharomyces cerevisiae

We identified and mapped RNA-binding sites of yeast Saccharomyces cerevisiae translation initiation factor eIF4G1 and examined their importance for eIF4G1 function in vitro and in vivo. Yeast eIF4G1 binds to single-stranded RNA with three different sites, the regions of amino acids 1-82 (N terminus), 492-539 (middle), and 883-952 (C terminus). The middle and C-terminal RNA-binding sites represent RS (arginine and serine)-rich domains; the N-terminal site is asparagine-, glutamine- and glycine-rich. The three RNA-binding sites have similar affinity for single-stranded RNA, whereas the affinity for single-stranded RNA full-length eIF4G1 is about 100-fold higher (approximate K(d) of 5 x 10(-8) M). Replacement of the arginine residues in the middle RS site by alanine residues abolishes its RNA-binding activity. Deletion of individual RNA-binding sites shows that eIF4G1 molecules lacking one binding site are still active in supporting growth of yeast cells and translation in vitro, whereas eIF4G1 molecules lacking two or all three RNA-binding sites are strongly impaired or inactive. These data suggest that RNA-binding activity is required for eIF4G1 function.     

Fragile X mental retardation protein (FMRP) binds specifically to the brain cytoplasmic RNAs BC1/BC200 via a novel RNA-binding motif

Fragile X mental retardation protein (FMRP), the protein responsible for the fragile X syndrome, is an RNA-binding protein involved in localization and translation of neuronal mRNAs. One of the RNAs known to interact with FMRP is the dendritic non-translatable brain cytoplasmic RNA 1 BC1 RNA that works as an adaptor molecule linking FMRP and some of its regulated mRNAs. Here, we showed that the N terminus of FMRP binds strongly and specifically to BC1 and to its potential human analog BC200. This region does not contain a motif known to specifically recognize RNA and thus constitutes a new RNA-binding motif. We further demonstrated that FMRP recognition involves the 5' stem loop of BC1 and that this is the region that exhibits complementarity to FMRP target mRNAs, raising the possibility that FMRP plays a direct role in BC1/mRNA annealing.     

Helicase associated 2 domain is essential for helicase activity of RNA helicase A

RNA helicase A (RHA), a DExD/H box protein, plays critical roles in a wide variety of cellular or viral functions. RHA contains a conserved core helicase domain that is flanked by five other domains. Two double-stranded RNA binding domains (dsRBD1 and dsRBD2) are at the N-terminus, whereas HA2 (helicase associated 2), OB-fold (oligonucleotide- or oligosaccharide-binding fold), and RGG (repeats of arginine and glycine–glycine residues) domains are at the C-terminus. The role of these domains in the helicase activity of RHA is still elusive due to the difficulty of obtaining enzymatically active mutant RHA. Here, we purified a series of mutant RHAs containing deletions in either N-terminus or C-terminus. Analysis of these mutant RHAs reveals that the dsRBDs are not required for RNA unwinding, but can enhance the helicase activity by promoting the binding of RHA to substrate RNA. In contrast, deletion of C-terminal domains including RGG, OB-fold, and HA2 does not significantly affect the binding of RHA to substrate RNA. However, HA2 is essential for the RNA unwinding by RHA whereas the RGG and OB-fold are dispensable. The results indicate that the core helicase domain alone is not enough for RHA to execute the unwinding activity.     

A small RNA activates CFA synthase by isoform‐specific mRNA stabilization

Small RNAs use a diversity of well‐characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq‐associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation‐independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot‐exposed, conserved 5′ end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E‐mediated decay in the 5′ untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA‐controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability.     

Translation initiation and the fate of bacterial mRNAs

Studies in pro- and eukaryotes have revealed that translation can determine the stability of a given messenger RNA. In bacteria, intrinsic mRNA signals can confer efficient ribosome binding, whereas translational feedback inhibition or environmental cues can interfere with this process. Such regulatory mechanisms are often controlled by RNA-binding proteins, small noncoding RNAs and structural rearrangements within the 5' untranslated region. Here, we review molecular events occurring in the 5' untranslated region of primarily Escherichia coli mRNAs with regard to their effects on mRNA stability.