Regulation of Leaderless mRNA Translation in Bacteria.

In bacteria, the translation of genetic information can begin through at least three different mechanisms: canonical or Shine-Dalgarno-led initiation, readthrough or 70S scanning initiation, or leaderless initiation. Here, we discuss the main features and regulation of the last, which is characterized mainly by the ability of 70S ribosomal particles to bind to AUG located at or near the 5' end of mRNAs to initiate translation. These leaderless mRNAs (lmRNAs) are rare in enterobacteria, such as Escherichia coli, but are common in other bacteria, such as Mycobacterium tuberculosis and Deinococcus deserti, where they may represent more than 20% and even up to 60% of the genes. Given that lmRNAs are devoid of a 5' untranslated region and the Shine-Dalgarno sequence located within it, the mechanism of translation regulation must depend on molecular strategies that are different from what has been observed in the Shine-Dalgarno-led translation. Diverse regulatory mechanisms have been proposed, including the processing of ribosomal RNA and changes in the abundance of translation factors, but all of them produce global changes in the initiation of lmRNA translation. Thus, further research will be required to understand how the initiation of the translation of particular lmRNA genes is regulated.

Upstream ORFs Influence Translation Efficiency in the Parasite Trypanosoma cruzi.

It is generally accepted that the presence of ORFs in the 5' untranslated region of eukaryotic transcripts modulates the production of proteins by controlling the translation initiation rate of the main CDS. In trypanosomatid parasites, which almost exclusively depend on post-transcriptional mechanisms to regulate gene expression, translation has been identified as a key step. However, the mechanisms of control of translation are not fully understood. In the present work, we have annotated the 5'UTRs of the Trypanosoma cruzi genome both in epimastigotes and metacyclic trypomastigotes and, using a stringent classification approach, we identified putative regulatory uORFs in about 9% of the analyzed 5'UTRs. The translation efficiency (TE) and translational levels of transcripts containing putative repressive uORFs were found to be significantly reduced. These findings are supported by the fact that proteomic methods only identify a low number of proteins coded by transcripts containing repressive uORF. We additionally show that AUG is the main translation initiator codon of repressive uORFs in T. cruzi. Interestingly, the decrease in TE is more pronounced when the uORFs overlaps the main CDS. In conclusion, we show that the presence of the uORF and features such as initiation codon and/or location of the uORFs may be acting to fine tune translation levels in these parasites.

Assessing the partners of the RBP9-mRNP complex in Trypanosoma cruzi using shotgun proteomics and RNA-seq.

Gene expression regulation in trypanosomes differs from other eukaryotes due to absence of transcriptional regulation for most of their genes. RNA-binding proteins (RBPs) associate with mRNAs and other regulatory proteins to form ribonucleoprotein complexes (mRNPs), which play a major role in post-transcriptional regulation. Here, we show that RBP9 is a cytoplasmic RBP in Trypanosoma cruzi with one RNA-recognition motif (RRM). The RBP9 sedimentation profile in a sucrose gradient indicated its presence in cytoplasmic translational complexes, suggesting its involvement in translation regulation. Taking this result as a motivation, we used shotgun proteomics and RNA-seq approaches to assess the core of the RBP9-mRNP complex. In epimastigotes in exponential growth, the complex was composed mostly by RBPs involved in RNA metabolism, such as ZC3H39, UBP1/2, NRBD1, and ALBA3/4. When parasites were subjected to nutritional stress, our analysis identified regulatory RBPs and the translation initiation factors eIF4E5, eIF4G5, eIF4G1, and eIF4G4. The RNA-seq results showed that RBP9-mRNP complex regulates transcripts encoding some RBPs - e.g. RBP5, RBP6, and RBP10 -, and proteins involved in metabolic processes. Therefore, we argue that RBP9 is part of cytoplasmic mRNPs complexes associated with mRNA metabolism and translation regulation in T. cruzi.

A Novel Function of Pet54 in Regulation of Cox1 Synthesis in Saccharomyces cerevisiae Mitochondria.

Cytochrome c oxidase assembly requires the synthesis of the mitochondria-encoded core subunits, Cox1, Cox2, and Cox3. In yeast, Pet54 protein is required to activate translation of the COX3 mRNA and to process the aI5ß intron on the COX1 transcript. Here we report a third, novel function of Pet54 on Cox1 synthesis. We observed that Pet54 is necessary to achieve an efficient Cox1 synthesis. Translation of the COX1 mRNA is coupled to the assembly of cytochrome c oxidase by a mechanism that involves Mss51. This protein activates translation of the COX1 mRNA by acting on the COX1 5'-UTR, and, in addition, it interacts with the newly synthesized Cox1 protein in high molecular weight complexes that include the factors Coa3 and Cox14. Deletion of Pet54 decreased Cox1 synthesis, and, in contrast to what is commonly observed for other assembly mutants, double deletion of cox14 or coa3 did not recover Cox1 synthesis. Our results show that Pet54 is a positive regulator of Cox1 synthesis that renders Mss51 competent as a translational activator of the COX1 mRNA and that this role is independent of the assembly feedback regulatory loop of Cox1 synthesis. Pet54 may play a role in Mss51 hemylation/conformational change necessary for translational activity. Moreover, Pet54 physically interacts with the COX1 mRNA, and this binding was independent of the presence of Mss51.