A novel class of microRNA-recognition elements that function only within open reading frames.

MicroRNAs (miRNAs) are well known to target 3' untranslated regions (3' UTRs) in mRNAs, thereby silencing gene expression at the post-transcriptional level. Multiple reports have also indicated the ability of miRNAs to target protein-coding sequences (CDS); however, miRNAs have been generally believed to function through similar mechanisms regardless of the locations of their sites of action. Here, we report a class of miRNA-recognition elements (MREs) that function exclusively in CDS regions. Through functional and mechanistic characterization of these 'unusual' MREs, we demonstrate that CDS-targeted miRNAs require extensive base-pairing at the 3' side rather than the 5' seed; cause gene silencing in an Argonaute-dependent but GW182-independent manner; and repress translation by inducing transient ribosome stalling instead of mRNA destabilization. These findings reveal distinct mechanisms and functional consequences of miRNAs that target CDS versus the 3' UTR and suggest that CDS-targeted miRNAs may use a translational quality-control-related mechanism to regulate translation in mammalian cells

The Dynamics of Supply and Demand in mRNA Translation

We study the elongation stage of mRNA translation in eukaryotes and find that, in contrast to the assumptions of previous models, both the supply and the demand for tRNA resources are important for determining elongation rates. We find that increasing the initiation rate of translation can lead to the depletion of some species of aa-tRNA, which in turn can lead to slow codons and queueing. Particularly striking “competition” effects are observed in simulations of multiple species of mRNA which are reliant on the same pool of tRNA resources. These simulations are based on a recent model of elongation which we use to study the translation of mRNA sequences from the Saccharomyces cerevisiae genome. This model includes the dynamics of the use and recharging of amino acid tRNA complexes, and we show via Monte Carlo simulation that this has a dramatic effect on the protein production behaviour of the system

Kaposi's Sarcoma-Associated Herpesvirus ORF57 Protein Binds and Protects a Nuclear Noncoding RNA from Cellular RNA Decay Pathways

The control of RNA stability is a key determinant in cellular gene expression. The stability of any transcript is modulated through the activity of cis- or trans-acting regulatory factors as well as cellular quality control systems that ensure the integrity of a transcript. As a result, invading viral pathogens must be able to subvert cellular RNA decay pathways capable of destroying viral transcripts. Here we report that the Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein binds to a unique KSHV polyadenylated nuclear RNA, called PAN RNA, and protects it from degradation by cellular factors. ORF57 increases PAN RNA levels and its effects are greatest on unstable alleles of PAN RNA. Kinetic analysis of transcription pulse assays shows that ORF57 protects PAN RNA from a rapid cellular RNA decay process, but ORF57 has little effect on transcription or PAN RNA localization based on chromatin immunoprecipitation and in situ hybridization experiments, respectively. Using a UV cross-linking technique, we further demonstrate that ORF57 binds PAN RNA directly in living cells and we show that binding correlates with function. In addition, we define an ORF57-responsive element (ORE) that is necessary for ORF57 binding to PAN RNA and sufficient to confer ORF57-response to a heterologous intronless β-globin mRNA, but not its spliced counterparts. We conclude that ORF57 binds to viral transcripts in the nucleus and protects them from a cellular RNA decay pathway. We propose that KSHV ORF57 protein functions to enhance the nuclear stability of intronless viral transcripts by protecting them from a cellular RNA quality control pathway

Expression of Human nPTB Is Limited by Extreme Suboptimal Codon Content

Background: The frequency of synonymous codon usage varies widely between organisms. Suboptimal codon content limits expression of viral, experimental or therapeutic heterologous proteins due to limiting cognate tRNAs. Codon content is therefore often adjusted to match codon bias of the host organism. Codon content also varies between genes within individual mammalian species. However, little attention has been paid to the consequences of codon content upon translation of host proteins. Methodology/Principal Findings: In comparing the splicing repressor activities of transfected human PTB and its two tissue-restricted paralogs–nPTB and ROD1–we found that the three proteins were expressed at widely varying levels. nPTB was expressed at 1–3 % the level of PTB despite similar levels of mRNA expression and 74 % amino acid identity. The low nPTB expression was due to the high proportion of codons with A or U at the third codon position, which are suboptimal in human mRNAs. Optimization of the nPTB codon content, akin to the ‘‘humanization’ ’ of foreign ORFs, allowed efficient translation in vivo and in vitro to levels comparable with PTB. We were then able to demonstrate that all three proteins act as splicing repressors. Conclusions/Significance: Our results provide a striking illustration of the importance of mRNA codon content in determining levels of protein expression, even within cells of the natural host species

The Leucine Zipper Domains of the Transcription Factors GCN4 and c-Jun Have Ribonuclease Activity

Basic-region leucine zipper (bZIP) proteins are one of the largest transcription factor families that regulate a wide range of cellular functions. Owing to the stability of their coiled coil structure leucine zipper (LZ) domains of bZIP factors are widely employed as dimerization motifs in protein engineering studies. In the course of one such study, the X-ray structure of the retro-version of the LZ moiety of yeast transcriptional activator GCN4 suggested that this retro-LZ may have ribonuclease activity. Here we show that not only the retro-LZ but also the authentic LZ of GCN4 has weak but distinct ribonuclease activity. The observed cleavage of RNA is unspecific, it is not suppressed by the ribonuclease A inhibitor RNasin and involves the breakage of 3′,5′-phosphodiester bonds with formation of 2′,3′-cyclic phosphates as the final products as demonstrated by HPLC/electrospray ionization mass spectrometry. Several mutants of the GCN4 leucine zipper are catalytically inactive, providing important negative controls and unequivocally associating the enzymatic activity with the peptide under study. The leucine zipper moiety of the human factor c-Jun as well as the entire c-Jun protein are also shown to catalyze degradation of RNA. The presented data, which was obtained in the test-tube experiments, adds GCN4 and c-Jun to the pool of proteins with multiple functions (also known as moonlighting proteins). If expressed in vivo, the endoribonuclease activity of these bZIP-containing factors may represent a direct coupling between transcription activation and controlled RNA turnover. As an additional result of this work, the retro-leucine zipper of GCN4 can be added to the list of functional retro-peptides

Coordinated Destruction of Cellular Messages in Translation Complexes by the Gammaherpesvirus Host Shutoff Factor and the Mammalian Exonuclease Xrn1

Several viruses encode factors that promote host mRNA degradation to silence gene expression. It is unclear, however, whether cellular mRNA turnover pathways are engaged to assist in this process. In Kaposi's sarcoma-associated herpesvirus this phenotype is enacted by the host shutoff factor SOX. Here we show that SOX-induced mRNA turnover is a two-step process, in which mRNAs are first cleaved internally by SOX itself then degraded by the cellular exonuclease Xrn1. SOX therefore bypasses the regulatory steps of deadenylation and decapping normally required for Xrn1 activation. SOX is likely recruited to translating mRNAs, as it cosediments with translation initiation complexes and depletes polysomes. Cleaved mRNA intermediates accumulate in the 40S fraction, indicating that recognition occurs at an early stage of translation. This is the first example of a viral protein commandeering cellular mRNA turnover pathways to destroy host mRNAs, and suggests that Xrn1 is poised to deplete messages undergoing translation in mammalian cells

The surprising negative correlation of gene length and optimal codon use - disentangling translational selection from GC-biased gene conversion in yeast

<p>Abstract</p> <p>Background</p> <p>Surprisingly, in several multi-cellular eukaryotes optimal codon use correlates negatively with gene length. This contrasts with the expectation under selection for translational accuracy. While suggested explanations focus on variation in strength and efficiency of translational selection, it has rarely been noticed that the negative correlation is reported only in organisms whose optimal codons are biased towards codons that end with G or C (-GC). This raises the question whether forces that affect base composition - such as GC-biased gene conversion - contribute to the negative correlation between optimal codon use and gene length.</p> <p>Results</p> <p>Yeast is a good organism to study this as equal numbers of optimal codons end in -GC and -AT and one may hence compare frequencies of optimal GC- with optimal AT-ending codons to disentangle the forces. Results of this study demonstrate in yeast frequencies of GC-ending (optimal AND non-optimal) codons decrease with gene length and increase with recombination. A decrease of GC-ending codons along genes contributes to the negative correlation with gene length. Correlations with recombination and gene expression differentiate between GC-ending and optimal codons, and also substitution patterns support effects of GC-biased gene conversion.</p> <p>Conclusion</p> <p>While the general effect of GC-biased gene conversion is well known, the negative correlation of optimal codon use with gene length has not been considered in this context before. Initiation of gene conversion events in promoter regions and the presence of a gene conversion gradient most likely explain the observed decrease of GC-ending codons with gene length and gene position.</p

SARS Coronavirus nsp1 Protein Induces Template-Dependent Endonucleolytic Cleavage of mRNAs: Viral mRNAs Are Resistant to nsp1-Induced RNA Cleavage

SARS coronavirus (SCoV) nonstructural protein (nsp) 1, a potent inhibitor of host gene expression, possesses a unique mode of action: it binds to 40S ribosomes to inactivate their translation functions and induces host mRNA degradation. Our previous study demonstrated that nsp1 induces RNA modification near the 5′-end of a reporter mRNA having a short 5′ untranslated region and RNA cleavage in the encephalomyocarditis virus internal ribosome entry site (IRES) region of a dicistronic RNA template, but not in those IRES elements from hepatitis C or cricket paralysis viruses. By using primarily cell-free, in vitro translation systems, the present study revealed that the nsp1 induced endonucleolytic RNA cleavage mainly near the 5′ untranslated region of capped mRNA templates. Experiments using dicistronic mRNAs carrying different IRESes showed that nsp1 induced endonucleolytic RNA cleavage within the ribosome loading region of type I and type II picornavirus IRES elements, but not that of classical swine fever virus IRES, which is characterized as a hepatitis C virus-like IRES. The nsp1-induced RNA cleavage of template mRNAs exhibited no apparent preference for a specific nucleotide sequence at the RNA cleavage sites. Remarkably, SCoV mRNAs, which have a 5′ cap structure and 3′ poly A tail like those of typical host mRNAs, were not susceptible to nsp1-mediated RNA cleavage and importantly, the presence of the 5′-end leader sequence protected the SCoV mRNAs from nsp1-induced endonucleolytic RNA cleavage. The escape of viral mRNAs from nsp1-induced RNA cleavage may be an important strategy by which the virus circumvents the action of nsp1 leading to the efficient accumulation of viral mRNAs and viral proteins during infection