Circular RNAs (circRNAs) are covalently closed, endogenous biomolecules in eukaryotes with tissue-specific and cell-specific expression patterns, whose biogenesis is regulated by specific cis-acting elements and trans-acting factors. Some circRNAs are abundant and evolutionarily conserved, and many circRNAs exert important biological functions by acting as microRNA or protein inhibitors ('sponges'), by regulating protein function or by being translated themselves. Furthermore, circRNAs have been implicated in diseases such as diabetes mellitus, neurological disorders, cardiovascular diseases and cancer. Although the circular nature of these transcripts makes their detection, quantification and functional characterization challenging, recent advances in high-throughput RNA sequencing and circRNA-specific computational tools have driven the development of state-of-the-art approaches for their identification, and novel approaches to functional characterization are emerging.

The biogenesis, biology and characterization of circular RNAs.

Dynamic RNA Modifications in Gene Expression Regulation

Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis

The recent discovery of reversible mRNA methylation has opened a new realm of post-transcriptional gene regulation in eukaryotes. The identification and functional characterization of proteins that specifically recognize RNA N6-methyladenosine (m6A) unveiled it as a modification that cells utilize to accelerate mRNA metabolism and translation. N6-adenosine methylation directs mRNAs to distinct fates by grouping them for differential processing, translation and decay in processes such as cell differentiation, embryonic development and stress responses. Other mRNA modifications, including N1-methyladenosine (m1A), 5-methylcytosine (m5C) and pseudouridine, together with m6A form the epitranscriptome and collectively code a new layer of information that controls protein synthesis.

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Post-transcriptional gene regulation by mRNA modifications

Extensive pre-mRNA back-splicing generates numerous circular RNAs (circRNAs) in human transcriptome. However, the biological functions of these circRNAs remain largely unclear. Here we report that N6-methyladenosine (m6A), the most abundant base modification of RNA, promotes efficient initiation of protein translation from circRNAs in human cells. We discover that consensus m6A motifs are enriched in circRNAs and a single m6A site is sufficient to drive translation initiation. This m6A-driven translation requires initiation factor eIF4G2 and m6A reader YTHDF3, and is enhanced by methyltransferase METTL3/14, inhibited by demethylase FTO, and upregulated upon heat shock. Further analyses through polysome profiling, computational prediction and mass spectrometry reveal that m6A-driven translation of circRNAs is widespread, with hundreds of endogenous circRNAs having translation potential. Our study expands the coding landscape of human transcriptome, and suggests a role of circRNA-derived proteins in cellular responses to environmental stress.

/pdf/extensive-translation-of-circular-rnas-driven-by-n-6-1lcpd9vw20.pdf

Extensive translation of circular RNAs driven by N 6 -methyladenosine

5′ UTR m6A Promotes Cap-Independent Translation

In eukaryotes, exposure to stress conditions causes a shift from cap-dependent to cap-independent translation. In trypanosomatids, environmental switches are the driving force of a developmental program of gene expression, but it is yet unclear how their translation machinery copes with their constantly changing environment. Trypanosomatids have a unique cap structure (cap-4) and encode four highly diverged paralogs of the cap-binding protein, eIF4E; none were found to genetically complement a yeast mutant failing to express eIF4E. Here we show that in promastigotes, a typical cap-binding complex is anchored through LeishIF4E-4, which associates with components of the cap-binding pre-initiation complex. In axenic amastigotes, expression of LeishIF4E-4 decreases and the protein does not bind the cap, whereas LeishIF4E-1 maintains its expression level and associates with the cap structure and with translation initiation factors. However, LeishIF4E-1 does not interact with eIF4G-like proteins in both life stages, excluding its involvement in cap-dependent translation. Using pull-down assays and mass-spectrometry, we identified a novel, non-conserved 4E-Interacting Protein (Leish4E-IP), which binds to LeishIF4E-1 in promastigotes, but not in amastigotes. Yeast two-hybrid and NMR spectroscopy confirmed the specificity of this interaction. We propose that Leish4E-IP is a translation regulator that is involved in switching between cap-dependent and alternative translation pathways.

A novel 4E-interacting protein in Leishmania is involved in stage-specific translation pathways.

Translation initiation in eukaryotes is mediated by assembly of the eIF4F complex over the m(7)GTP cap structure at the 5'-end of mRNAs. This requires an interaction between eIF4E and eIF4G, two eIF4F subunits. The Leishmania orthologs of eIF4E are structurally diverged from their higher eukaryote counterparts, since they have evolved to bind the unique trypanosomatid cap-4 structure. Here, we characterize a key eIF4G candidate from Leishmania parasites (LeishIF4G-3) that contains a conserved MIF4G domain. LeishIF4G-3 was found to coelute with the parasite eIF4F subunits from an m(7)GTP-Sepharose column and to bind directly to LeishIF4E. In higher eukaryotes the eIF4E-eIF4G interaction is based on a conserved peptide signature [Y(X(4))Lphi], where X is any amino acid and Phi is a hydrophobic residue. A parallel eIF4E-binding peptide was identified in LeishIF4G-3 (20-YPGFSLDE-27). However, the binding motif varies extensively: in addition to Y20 and L25, binding strictly requires the presence of F23, whereas the hydrophobic amino acid (Phi) is dispensable. The LeishIF4E-LeishIF4G-3 interaction was also confirmed by nuclear magnetic resonance (NMR) studies. In view of these diversities, the characterization of the parasite eIF4E-eIF4G interaction may not only serve as a novel target for inhibiting Leishmaniasis but also provide important insight for future drug discovery.

/pdf/evolutionary-changes-in-the-leishmania-eif4f-complex-involve-q5ny18hlva.pdf

Alexandra Zinoviev

Papers

5′ UTR m6A Promotes Cap-Independent Translation

A novel 4E-interacting protein in Leishmania is involved in stage-specific translation pathways.

Evolutionary changes in the Leishmania eIF4F complex involve variations in the eIF4E–eIF4G interactions

Release of Ubiquitinated and Non-ubiquitinated Nascent Chains from Stalled Mammalian Ribosomal Complexes by ANKZF1 and Ptrh1.

Multiple Mechanisms of Reinitiation on Bicistronic Calicivirus mRNAs