Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity

RNase E plays an essential role in RNA processing and decay and tethers to the cytoplasmic membrane in Escherichia coli; however, the function of this membrane-protein interaction has remained unclear. Here, we establish a mechanistic role for the RNase E-membrane interaction. The reconstituted highly conserved N-terminal fragment of RNase E (NRne, residues 1-499) binds specifically to anionic phospholipids through electrostatic interactions. The membrane-binding specificity of NRne was confirmed using circular dichroism difference spectroscopy; the dissociation constant (K(d)) for NRne binding to anionic liposomes was 298 nM. E. coli RNase G and RNase E/G homologs from phylogenetically distant Aquifex aeolicus, Haemophilus influenzae Rd, and Synechocystis sp. were found to be membrane-binding proteins. Electrostatic potentials of NRne and its homologs were found to be conserved, highly positive, and spread over a large surface area encompassing four putative membrane-binding regions identified in the "large" domain (amino acids 1-400, consisting of the RNase H, S1, 5'-sensor, and DNase I subdomains) of E. coli NRne. In vitro cleavage assay using liposome-free and liposome-bound NRne and RNA substrates BR13 and GGG-RNAI showed that NRne membrane binding altered its enzymatic activity. Circular dichroism spectroscopy showed no obvious thermotropic structural changes in membrane-bound NRne between 10 and 60 °C, and membrane-bound NRne retained its normal cleavage activity after cooling. Thus, NRne membrane binding induced changes in secondary protein structure and enzymatic activation by stabilizing the protein-folding state and increasing its binding affinity for its substrate. Our results demonstrate that RNase E-membrane interaction enhances the rate of RNA processing and decay.     

Cup is a nucleocytoplasmic shuttling protein that interacts with the eukaryotic translation initiation factor 4E to modulate Drosophila ovary development

In Drosophila, the product of the fs (2)cup gene (Cup) is known to be crucial for diverse aspects of female germ-line development. Its functions at the molecular level, however, have remained mainly unexplored. Cup was found to directly associate with eukaryotic translation initiation factor 4E (eIF4E). In this report, we show that Cup is a nucleocytoplasmic shuttling protein and that the interaction with eIF4E promotes retention of the Cup protein in the cytoplasm. Cup is required for the correct accumulation and localization of eIF4E within the posterior cytoplasm of developing oocytes. We furthermore show that cup and eIF4E interact genetically, because a reduction in the level of eIF4E activity deteriorates the development and growth of ovaries bearing homozygous cup mutant alleles. Our results reveal a crucial role for the Cup-eIF4E complex in ovary-specific developmental programs.     

Small RNA binding to the lateral surface of Hfq hexamers and structural rearrangements upon mRNA target recognition

The bacterial Sm-like protein Hfq is a central player in the control of bacterial gene expression. Hfq forms complexes with small regulatory RNAs (sRNAs) that use complementary "seed" sequences to target specific mRNAs. Hfq forms hexameric rings, which preferably bind uridine-rich RNA 3' ends on their proximal surface and adenine-rich sequences on their distal surface. However, many reported properties of Hfq/sRNA complexes could not be explained by these RNA binding modes. Here, we use the RybB sRNA to identify the lateral surface of Hfq as a third, independent RNA binding surface. A systematic mutational analysis and competition experiments demonstrate that the lateral sites have a preference for and are sufficient to bind the sRNA "body," including the seed sequence. Furthermore, we detect significant structural rearrangements of the Hfq/sRNA complex upon mRNA target recognition that lead to a release of the seed sequence, or of the entire sRNA molecule in case of an unfavorable 3' end. Consequently, we propose a molecular model for the Hfq/sRNA complex, where the sRNA 3' end is anchored in the proximal site of Hfq, whereas the sRNA body, including the seed sequence, is bound by up to six of the lateral sites. In contrast to previously proposed arrangements, the presented model explains how Hfq can protect large parts of the sRNA body while still allowing a rapid recycling of sRNAs. Furthermore, our model suggests molecular mechanisms for the function of Hfq as an RNA chaperone and for the molecular events that are initiated upon mRNA target recognition.     

70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

According to the standard model of bacterial translation initiation, the small ribosomal 30S subunit binds to the initiation site of an mRNA with the help of three initiation factors (IF1–IF3). Here, we describe a novel type of initiation termed “70S-scanning initiation,” where the 70S ribosome does not necessarily dissociate after translation of a cistron, but rather scans to the initiation site of the downstream cistron. We detailed the mechanism of 70S-scanning initiation by designing unique monocistronic and polycistronic mRNAs harboring translation reporters, and by reconstituting systems to characterize each distinct mode of initiation. Results show that 70S scanning is triggered by fMet-tRNA and does not require energy; the Shine–Dalgarno sequence is an essential recognition element of the initiation site. IF1 and IF3 requirements for the various initiation modes were assessed by the formation of productive initiation complexes leading to synthesis of active proteins. IF3 is essential and IF1 is highly stimulating for the 70S-scanning mode. The task of IF1 appears to be the prevention of untimely interference by ternary aminoacyl (aa)-tRNA•elongation factor thermo unstable (EF-Tu)•GTP complexes. Evidence indicates that at least 50% of bacterial initiation events use the 70S-scanning mode, underscoring the relative importance of this translation initiation mechanism.It is textbook knowledge that 30S subunits initiate protein synthesis in bacteria; they recognize the initiation site of the mRNA composed of the Shine–Dalgarno (SD) sequence, the AUG codon, and fMet-tRNA, together with three initiation factors (IFs) forming the 30S initiation complex (30SIC). Association of the large 50S subunit triggers the release of the IFs, leading to the 70S initiation complex (70SIC) that enters the elongation phase of translation (reviewed in 1). We term this initiation path the “30S-binding mode” of bacterial initiation. After elongation and termination, it is thought that the ribosome dissociates into its subunits, thus providing 30S subunits for the next round of initiation.The functional role of IF2 is well defined. It can bind directly to the 30S, providing a docking site for fMet-tRNA (2), but it can also enter the 30S subunit as ternary complex fMet-tRNA•IF2•GTP (3). Both IF2 and IF3 are essential for viability. IF3 has a binding site at the 30S interface (4), which explains its antiassociation effect (5, 6), as well as its role in dissociation of the terminating 70S ribosome (7). However, the in vivo concentration of IF3 is 100-fold less (8) than required for full dissociation of 70S in vitro (4). Evidence for the presence of IF3 on 70S ribosomes was reported (9), indicating that the functional spectrum of IF3 is possibly not restricted to an antiassociation effect. Both IF3 and IF2 are also responsible for the fidelity of decoding the initiation AUG by fMet-tRNAfMet at the P site of 30S subunits (10).IF1 is universal (11) and essential for viability (12). It is the smallest factor, with 72 amino acid residues in Escherichia coli, and binds to the decoding center at the ribosomal A site (13). Several functions have been described, including stimulation of the formation of the 30SIC and subunit association (14). Interference with the binding of ternary complexes aminoacyl (aa)-tRNA•elongation factor thermo unstable (EF-Tu)•GTP to 30S subunits has also been suggested (15). Omitting IF1 in 30S-binding tests decreased the accuracy of fMet-tRNA selection over the elongator Phe-tRNA about 60-fold, which was suggested to account for the essential nature of IF1 (16). All three factors are thought to dissociate upon 50S arrival or shortly thereafter (1). IF1 is required for proper initiator-tRNA selection on 70S along with IF2 and IF3, in contrast to the 30SIC, where IF2 and IF3 provide tRNA selection (17).In addition to the 30S-binding initiation, a second initiation mode exists that has a niche existence in bacteria: Leaderless mRNA (lmRNA) contains an initiator AUG codon within the first 5 nt at the 5′-end, and thus does not contain an SD sequence. This initiation mode uses 70S ribosomes with the special feature that the ribosomal proteins S1 and S2 are not required, which are otherwise important for the 30S-binding mode (18). Initiation of lmRNA can even occur in the absence of all IFs (19, 20). Additional information about lmRNAs is provided in SI Appendix, Introduction.The existence of a third initiation mode, viz. a 70S type of bacterial initiation, has been conjectured several times previously (21–23), although no in-depth mechanistic evidence has verified this mode thus far. For example:i) The formylation of the initiator Met-tRNAfMet in bacteria was interpreted as an indication of a 70S initiation mode (22). Indeed, only the anticodon loop of a tRNA and a part of the anticodon stem interact with the 30S subunit (24, 25), leaving the fMet residue as a substrate for the peptidyltransferase center on the large subunit within the 70S ribosome.ii) When an AUG codon without a preceding SD sequence follows a stop codon within a distance of <20 nt, a mutational study unexpectedly revealed that efficient protein synthesis can be initiated in vivo at this AUG codon. The interpretation was that ribosomes were sliding down from the stop codon of the preceding cistron, although it was not analyzed whether 70S ribosomes or 30S subunits were involved in sliding or whether factors were required (26). Further evidence for a 70S type of initiation is described in SI Appendix, Introduction and concerns both studies of translational coupling and a consideration of the fact that more than 75% of the intercistronic distances are shorter than 30 nt, which is too short to allow an independent termination of cistron n and initiation of downstream cistron n + 1 (SI Appendix, Fig. S1 A and B).Here, we demonstrate that there is an additional and frequent initiation mode that we term “70S-scanning initiation.” The 70S ribosomes, rather than the 30S subunits, scan the sequence surrounding the termination signal for the presence of an SD sequence after termination. Furthermore, we show that the requirement of IF1 and IF3 for the three initiation modes (30S binding, 70S scanning, and initiation of lmRNAs) is distinct for each mode.     

A 5′ cytosine binding pocket in Puf3p specifies regulation of mitochondrial mRNAs

A single regulatory protein can control the fate of many mRNAs with related functions. The Puf3 protein of Saccharomyces cerevisiae is exemplary, as it binds and regulates more than 100 mRNAs that encode proteins with mitochondrial function. Here we elucidate the structural basis of that specificity. To do so, we explore the crystal structures of Puf3p complexes with 2 cognate RNAs. The key determinant of Puf3p specificity is an unusual interaction between a distinctive pocket of the protein with an RNA base outside the “core” PUF-binding site. That interaction dramatically affects binding affinity in vitro and is required for regulation in vivo. The Puf3p structures, combined with those of Puf4p in the same organism, illuminate the structural basis of natural PUF-RNA networks. Yeast Puf3p binds its own RNAs because they possess a −2C and is excluded from those of Puf4p which contain an additional nucleotide in the core-binding site.     

DNA damage and eIF4G1 in breast cancer cells reprogram translation for survival and DNA repair mRNAs

The cellular response to DNA damage is mediated through multiple pathways that regulate and coordinate DNA repair, cell cycle arrest, and cell death. We show that the DNA damage response (DDR) induced by ionizing radiation (IR) is coordinated in breast cancer cells by selective mRNA translation mediated by high levels of translation initiation factor eIF4G1 (eukaryotic initiation factor 4γ1). Increased expression of eIF4G1, common in breast cancers, was found to selectively increase translation of mRNAs involved in cell survival and the DDR, preventing autophagy and apoptosis [Survivin, hypoxia inducible factor 1α (HIF1α), X-linked inhibitor of apoptosis (XIAP)], promoting cell cycle arrest [growth arrest and DNA damage protein 45a (GADD45a), protein 53 (p53), ATR-interacting protein (ATRIP), Check point kinase 1 (Chk1)] and DNA repair [p53 binding protein 1 (53BP1), breast cancer associated proteins 1, 2 (BRCA1/2), Poly-ADP ribose polymerase (PARP), replication factor c2–5 (Rfc2-5), ataxia telangiectasia mutated gene 1 (ATM), meiotic recombination protein 11 (MRE-11), and others]. Reduced expression of eIF4G1, but not its homolog eIF4G2, greatly sensitizes cells to DNA damage by IR, induces cell death by both apoptosis and autophagy, and significantly delays resolution of DNA damage foci with little reduction of overall protein synthesis. Although some mRNAs selectively translated by higher levels of eIF4G1 were found to use internal ribosome entry site (IRES)-mediated alternate translation, most do not. The latter group shows significantly reduced dependence on eIF4E for translation, facilitated by an enhanced requirement for eIF4G1. Increased expression of eIF4G1 therefore promotes specialized translation of survival, growth arrest, and DDR mRNAs that are important in cell survival and DNA repair following genotoxic DNA damage.     

Analysis of Chlamydomonas thiamin metabolism in vivo reveals riboswitch plasticity

Thiamin (vitamin B₁) is an essential micronutrient needed as a cofactor for many central metabolic enzymes. Animals must have thiamin in their diet, whereas bacteria, fungi, and plants can biosynthesize it de novo from the condensation of a thiazole and a pyrimidine moiety. Although the routes to biosynthesize these two heterocycles are not conserved in different organisms, in all cases exogenous thiamin represses expression of one or more of the biosynthetic pathway genes. One important mechanism for this control is via thiamin-pyrophosphate (TPP) riboswitches, regions of the mRNA to which TPP can bind directly, thus facilitating fine-tuning to maintain homeostasis. However, there is little information on how modulation of riboswitches affects thiamin metabolism in vivo. Here we use the green alga, Chlamydomonas reinhardtii, which regulates both thiazole and pyrimidine biosynthesis with riboswitches in the THI4 (Thiamin 4) and THIC (Thiamin C) genes, respectively, to investigate this question. Our study reveals that regulation of thiamin metabolism is not the simple dogma of negative feedback control. Specifically, balancing the provision of both of the heterocycles of TPP appears to be an important requirement. Furthermore, we show that the Chlamydomonas THIC riboswitch is controlled by hydroxymethylpyrimidine pyrophosphate, as well as TPP, but with an identical alternative splicing mechanism. Similarly, the THI4 gene is responsive to thiazole. The study not only provides insight into the plasticity of the TPP riboswitches but also shows that their maintenance is likely to be a consequence of evolutionary need as a function of the organisms’ environment and the particular pathway used.     

Human cytomegalovirus UL69 protein facilitates translation by associating with the mRNA cap-binding complex and excluding 4EBP1

4EBP1 is phosphorylated by the mTORC1 kinase. When mTORC1 activity is inhibited, hypophosphorylated 4EBP1 binds and sequesters eIF4E, a component of the mRNA cap-binding complex, and blocks translation. As a consequence, mTORC1 activity is needed to maintain active translation. The human cytomegalovirus pUL38 protein preserves mTORC1 activity, keeping most of the E4BP1 in the infected cell in a hyperphosphorylated, inactive state. Here we report that a second viral protein, pUL69, also antagonizes the activity of 4EBP1, but by a separate mechanism. pUL69 interacts directly with eIF4A1, an element of the cap-binding complex, and the poly(A)-binding protein, which binds to the complex. When pUL69 accumulates during infection with wild-type virus, 4EBP1 is excluded from the complex. However, 4EBP1 is present in the cap-binding complex after infection with a pUL69-deficient virus, coincident with reduced accumulation of several late virus-coded proteins. We propose that pUL69 supports translation in human cytomegalovirus-infected cells by excluding hypophosphorylated 4EBP1 from the cap-binding complex.     

An intermolecular base triple as the basis of ligand specificity and affinity in the guanine- and adenine-sensing riboswitch RNAs

Riboswitches are highly structured RNA elements that control the expression of many bacterial genes by binding directly to small metabolite molecules with high specificity and affinity. In Bacillus subtilis, two classes of riboswitches have been described that discriminate between guanine and adenine despite an extremely high degree of homology both in their primary and secondary structure. We have identified intermolecular base triples between both purine ligands and their respective riboswitch RNAs by NMR spectroscopy. Here, specificity is mediated by the formation of a Watson-Crick base pair between the guanine ligand and a C residue or the adenine ligand and a U residue of the cognate riboswitch RNA, respectively. In addition, a second base-pairing interaction common to both riboswitch purine complexes involves a uridine residue of the RNA and the N3/N9 edge of the purine ligands. This base pairing is mediated by a previously undescribed hydrogen-bonding scheme that contributes to the affinity of the RNA-ligand interaction. The observed intermolecular hydrogen bonds between the purine ligands and the RNA rationalize the previously observed change in specificity upon a C to U mutation in the core of the purine riboswitch RNAs and the differences in the binding affinities for a number of purine analogs.     

miR-519 reduces cell proliferation by lowering RNA-binding protein HuR levels

Gene expression is potently regulated through the action of RNA-binding proteins (RBPs) and microRNAs (miRNAs). Here, we present evidence of a miRNA regulating an RBP. The RBP HuR can stabilize and modulate the translation of numerous target mRNAs involved in cell proliferation, but little is known about the mechanisms that regulate HuR abundance. We identified two putative sites of miR-519 interaction on the HuR mRNA, one in its coding region (CR), one in its 3′-untranslated region (UTR). In several human carcinoma cell lines tested, HeLa (cervical), HCT116 and RKO (colon), and A2780 (ovarian), overexpression of a miR-519 precursor [(Pre)miR-519] reduced HuR abundance, while inhibiting miR-519 by using an antisense RNA [(AS)miR-519] elevated HuR levels. The influence of miR-519 was recapitulated using heterologous reporter constructs that revealed a greater repressive effect on the HuR CR than the HuR 3′-UTR target sequences. miR-519 did not alter HuR mRNA abundance, but reduced HuR biosynthesis, as determined by measuring nascent HuR translation and HuR mRNA association with polysomes. Modulation of miR-519 leading to altered HuR levels in turn affected the levels of proteins encoded by HuR target mRNAs. In keeping with HuR's proliferative influence, (AS)miR-519 significantly increased cell number and [³H]-thymidine incorporation, while (Pre)miR-519 reduced these parameters. Importantly, the growth-promoting effects of (AS)miR-519 required the presence of HuR, because downregulation of HuR by RNAi dramatically suppressed its proliferative action. In sum, miR-519 represses HuR translation, in turn reducing HuR-regulated gene expression and cell division.     

Selection of the mRNA translation initiation region by Escherichia coli ribosomes.

Two genes specifying model mRNAs of minimal size and coding capacity, with or without the Shine-Dalgarno (SD) sequence, were assembled, cloned, and transcribed in high yields. These mRNAs, as well as synthetic polynucleotides, phage MS2 RNA, and a deoxyoctanucleotide complementary to the 3' end of 16S rRNA were used to study the mechanism of translation initiation in vitro. Escherichia coli 30S ribosomal subunits interact with all these nucleic acids, albeit with different affinities; the affinity for the mRNA with the SD sequence (Ka approximately 2 x 10(7) M-1) is more than an order of magnitude higher than that for the mRNA lacking this sequence. The initiation factors are equally required, regardless of the presence of the SD sequence, for 30S and 70S initiation complex formation and for mRNA translation, but the initiation factors do not affect the SD interaction or the binding of the mRNAs to the ribosomes. The SD interaction is also mechanistically irrelevant for 30S initiation complex formation and is not essential for translation in vitro or for the selection of the mRNA reading frame. It is suggested that the function of the SD interaction is to ensure a high concentration of the initiation triplet near the ribosomal peptidyl-tRNA binding site, whereas the selection of the translational start is achieved kinetically, under the influence of the initiation factors, during decoding of the initiator tRNA.     

Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1–3 and 7–8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4–6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.