Local absence of secondary structure permits translation of mRNAs that lack ribosome-binding sites

The initiation of translation is a fundamental and highly regulated process in gene expression. Translation initiation in prokaryotic systems usually requires interaction between the ribosome and an mRNA sequence upstream of the initiation codon, the so-called ribosome-binding site (Shine-Dalgarno sequence). However, a large number of genes do not possess Shine-Dalgarno sequences, and it is unknown how start codon recognition occurs in these mRNAs. We have performed genome-wide searches in various groups of prokaryotes in order to identify sequence elements and/or RNA secondary structural motifs that could mediate translation initiation in mRNAs lacking Shine-Dalgarno sequences. We find that mRNAs without a Shine-Dalgarno sequence are generally less structured in their translation initiation region and show a minimum of mRNA folding at the start codon. Using reporter gene constructs in bacteria, we also provide experimental support for local RNA unfoldedness determining start codon recognition in Shine-Dalgarno--independent translation. Consistent with this, we show that AUG start codons reside in single-stranded regions, whereas internal AUG codons are usually in structured regions of the mRNA. Taken together, our bioinformatics analyses and experimental data suggest that local absence of RNA secondary structure is necessary and sufficient to initiate Shine-Dalgarno--independent translation. Thus, our results provide a plausible mechanism for how the correct translation initiation site is recognized in the absence of a ribosome-binding site.     

mRNAs containing the unstructured 5' leader sequence of alfalfa mosaic virus RNA 4 translate inefficiently in lysates from poliovirus-infected HeLa cells.

Poliovirus infection is accompanied by translational control that precludes translation of 5'-capped mRNAs and facilitates translation of the uncapped poliovirus RNA by an internal initiation mechanism. Previous reports have suggested that the capped alfalfa mosaic virus coat protein mRNA (AIMV CP RNA), which contains an unstructured 5' leader sequence, is unusual in being functionally active in extracts prepared from poliovirus-infected HeLa cells (PI-extracts). To identify the cis-acting nucleotide elements permitting selective AIMV CP expression, we tested capped mRNAs containing structured or unstructured 5' leader sequences in addition to an mRNA containing the poliovirus internal ribosome entry site (IRES). Translations were performed with PI-extracts and extracts prepared from mock-infected HeLa cells (MI-extracts). A number of control criteria demonstrated that the HeLa cells were infected by poliovirus and that the extracts were translationally active. The data strongly indicate that translation of RNAs lacking an internal ribosome entry site, including AIMV CP RNA, was severely compromised in PI-extracts, and we find no evidence that the unstructured AIMV CP RNA 5' leader sequence acts in cis to bypass the poliovirus translational control. Nevertheless, cotranslation assays in the MI-extracts demonstrate that mRNAs containing the unstructured AIMV CP RNA 5' untranslated region have a competitive advantage over those containing the rabbit alpha-globin 5' leader. Previous reports of AIMV CP RNA translation in PI-extracts likely describe inefficient expression that can be explained by residual cap-dependent initiation events, where AIMV CP RNA translation is competitive because of a diminished quantitative requirement for initiation factors.     

Effects of different target sites on antisense RNA-mediated regulation of gene expression

Antisense RNA is a type of noncoding RNA (ncRNA) that binds to complementary mRNA sequences and induces gene repression by inhibiting translation or degrading mRNA. Recently, several small ncRNAs (sRNAs) have been identified in Escherichia coli that act as antisense RNA mainly via base pairing with mRNA. The base pairing predominantly leads to gene repression, and in some cases, gene activation. In the current study, we examined how the location of target sites affects sRNA-mediated gene regulation. An efficient antisense RNA expression system was developed, and the effects of antisense RNAs on various target sites in a model mRNA were examined. The target sites of antisense RNAs suppressing gene expression were identified, not only in the translation initiation region (TIR) of mRNA, but also at the junction between the coding region and 3'' untranslated region. Surprisingly, an antisense RNA recognizing the upstream region of TIR enhanced gene expression through increasing mRNA stability. [BMB Reports 2014; 47(11): 619-624]     

JKTBP1 is involved in stabilization and IRES-dependent translation of NRF mRNAs by binding to 5' and 3' untranslated regions

Heterogeneous nuclear ribonucleoprotein D-like protein (JKTBP) 1 was implicated in cap-independent translation by binding to the internal ribosome entry site in the 5′ untranslated region (UTR) of NF-κB-repressing factor (NRF). Two different NRF mRNAs have been identified so far, both sharing the common 5′ internal ribosome entry site but having different length of 3′ UTRs. Here, we used a series of DNA and RNA luciferase reporter constructs comprising 5′, 3′ or both NRF UTRs to study the effect of JKTBP1 on translation of NRF mRNA variants. The results indicate that JKTBP1 regulates the level of NRF protein expression by binding to both NRF 5′ and 3′ UTRs. Using successive deletion and point mutations as well as RNA binding studies, we define two distinct JKTBP1 binding elements in NRF 5′ and 3′ UTRs. Furthermore, JKTBP1 requires two distinct RNA binding domains to interact with NRF UTRs and a short C-terminal region for its effect on NRF expression. Together, our study shows that JKTBP1 contributes to NRF protein expression via two disparate mechanisms: mRNA stabilization and cap-independent translation. By binding to 5′ UTR, JKTBP1 increases the internal translation initiation in both NRF mRNA variants, whereas its binding to 3′ UTR elevated primarily the stability of the major NRF mRNA. Thus, JKTBP1 is a key regulatory factor linking two pivotal control mechanisms of NRF gene expression: the cap-independent translation initiation and mRNA stabilization.     

真核mRNA的3′非翻译区转录后水平调控作用研究进展

真核生物mRNA的3′非翻译区(3′-UTR)在基因表达的转录后调控中起着重要作用:3′-UTR内存在末端加工信号以指导mRNA3′末端的加工;3′-UTR不但控制mRNA的稳定性及降解速率、协助辨认特殊密码子,而且还控制着mRNA的翻译时间、位点及控制其翻译起始及效率等。     

Alternative translation of human fibroblast growth factor 2 mRNA occurs by internal entry of ribosomes.

Alternative initiations of translation of the human fibroblast growth factor 2 (FGF-2) mRNA, at three CUG start codons and one AUG start codon, result in the synthesis of four isoforms of FGF-2. This process has important consequences on the fate of FGF-2: the CUG-initiated products are nuclear and their constitutive expression is able to induce cell immortalization, whereas the AUG-initiated product, mostly cytoplasmic, can generate cell transformation. Thus, the different isoforms probably have distinct targets in the cell. We show here that translation initiation of the FGF-2 mRNA breaks the rule of the cap-dependent ribosome scanning mechanism. First, translation of the FGF-2 mRNA was shown to be cap independent in vitro. This cap-independent translation required a sequence located between nucleotides (nt) 192 and 256 from the 5' end of the 318-nt-long 5' untranslated region. Second, expression of bicistronic vectors in COS-7 cells indicated that the FGF-2 mRNA is translated through a process of internal ribosome entry mediated by the mRNA leader sequence. By introducing additional AUG codons into the RNA leader sequence, we localized an internal ribosome entry site to between nt 154 and 318 of the 5' untranslated region, just upstream of the first CUG. The presence of an internal ribosome entry site in the FGF-2 mRNA suggests that the process of internal translation initiation, by controlling the expression of a growth factor, could have a crucial role in the control of cell proliferation and differentiation.     

Translational regulation by mRNA/protein interactions in eukaryotic cells: Ferritin and beyond

The expression of certain eukaryotic genes is – at least in part – controlled at the level of mRNA translation. The step of translational initiation represents the primary target for regulation. The regulation of the intracellular iron storage protein ferritin in response to iron levels provides a good example of translational control by a reversible RNA/protein interaction in the 5' untranslated region of an mRNA. We consider mechanisms by which mRNA/protein interactions may impede translation initiation and discuss recent data suggesting that the ferritin example may represent the ‘tip of the iceberg’ of a more general theme for translational control.     

The influence of mRNA primary and secondary structure on human IFN-gamma gene expression in E. coli.

Parameters influencing the efficiency of expression of the human immune interferon (IFN-gamma) gene in E. coli were studied by comparing a series of eight in vitro-derived gene variants. These contained all possible combinations of silent mutations in the first three codons of the mature IFN-gamma polypeptide coding sequence. Expression levels varied up to 50-fold among the different constructions. Comparison of messenger RNA secondary structure models for each variant suggested that the presence of stem-loop structures blocking the translation initiation signals could drastically decrease the efficiency of IFN-gamma synthesis. With variants displaying no stable mRNA secondary structure in the region, a C----U transition at position +11 after the AUG resulted in a 5-fold increase in expression indicating that RNA primary structure also plays an important role in expression. In addition we demonstrate that, in this system, a spacing of 8 nucleotides between the Shine-Dalgarno region and AUG was optimal for gene expression and that the steady-state production level of IFN-gamma rose exponentially with increasing rate of synthesis.     

Mutations affecting pseudoknot control of the replication of B group plasmids.

The translational initiation region of the mRNA for the replication initiation protein (RepA) of pMU720 is predicted to be sequestered in an inhibitory secondary structure designated stem-loop III. Activation of repA translation requires both the disruption of stem-loop III by ribosomes involved in the translation and termination of the leader peptide RepB and the formation of a pseudoknot, a tertiary RNA structure. Disruption of stem-loop III by site-directed mutagenesis was found to be insufficient to allow high repA expression in the absence of pseudoknot formation, indicating that the pseudoknot acts as an enhancer of repA translation. Furthermore, extending the length of the leader peptide RepB and changing the distance between the pseudoknot and repA Shine-Dalgarno sequence were found to have major effects on the translation of repA.     

A sequence required for -1 ribosomal frameshifting located four kilobases downstream of the frameshift site

Programmed ribosomal frameshifting allows one mRNA to encode regulate expression of, multiple open reading frames (ORFs). The polymerase encoded by ORF 2 of Barley yellow dwarf virus (BYDV) is expressed via minus one (-1) frameshifting from the overlapping ORF 1. Previously, this appeared to be mediated by a 116 nt RNA sequence that contains canonical -1 frameshift signals including a shifty heptanucleotide followed by a highly structured region. However, unlike known -1 frameshift signals, the reporter system required the zero frame stop codon and did not require a consensus shifty site for expression of the -1 ORF. In contrast, full-length viral RNA required a functional shifty site for frameshifting in wheat germ extract, while the stop codon was not required. Increasing translation initiation efficiency by addition of a 5' cap on the naturally uncapped viral RNA, decreased the frameshift rate. Unlike any other known RNA, a region four kilobases downstream of the frameshift site was required for frameshifting. This included an essential 55 base tract followed by a 179 base tract that contributed to full frameshifting. The effects of most mutations on frameshifting correlated with the ability of viral RNA to replicate in oat protoplasts, indicating that the wheat germ extract accurately reflected control of BYDV RNA translation in the infected cell. However, the overall frameshift rate appeared to be higher in infected cells, based on immunodetection of viral proteins. These findings show that use of short recoding sequences out of context in reporter constructs may overlook distant signals. Most importantly, the remarkably long-distance interaction reported here suggests the presence of a novel structure that can facilitate ribosomal frameshifting.     

Translation initiation factor-dependent extracts from Saccharomyces cerevisiae

Translation initiation factor 4A- and 4E-dependent extracts were developed from Saccharomyces cerevisiae and used to study factor requirements for translation of individual mRNAs in vitro. Whereas all mRNAs tested required eIF-4A, mRNAs devoid of secondary structure in their 5' untranslated region did not require exogenous eIF-4E for translation. The latter included alfalfa mosaic virus RNA4, mRNA containing the untranslated region of tobacco mosaic virus RNA and mRNA containing part of the untranslated region of poliovirus RNA. Furthermore, initiation of translation on mRNAs containing part of the untranslated region of poliovirus RNA is most likely internal.     

RNA Recognition Motif 2 of Yeast Pab1p Is Required for Its Functional Interaction with Eukaryotic Translation Initiation Factor 4G

The eukaryotic mRNA 3′ poly(A) tail and its associated poly(A)-binding protein (Pab1p) are important regulators of gene expression. One role for this complex in the yeast Saccharomyces cerevisiae is in translation initiation through an interaction with a 115-amino-acid region of the translation initiation factor eIF4G. The eIF4G-interacting domain of Pab1p was mapped to its second RNA recognition motif (RRM2) in an in vitro binding assay. Moreover, RRM2 of Pab1p was required for poly(A) tail-dependent translation in yeast extracts. An analysis of a site-directed Pab1p mutation which bound to eIF4G but did not stimulate translation of uncapped, polyadenylated mRNA suggested additional Pab1p-dependent events during translation initiation. These results support the model that the association of RRM2 of yeast Pab1p with eIF4G is a prerequisite for the poly(A) tail to stimulate the translation of mRNA in vitro.