Readers of the m6A epitranscriptomic code

International audienceN6-methyl adenosine (m6A) is the most prevalent and evolutionarily conserved, modification of polymerase II transcribed RNAs. By post-transcriptionally controlling patterns of gene expression, m6A deposition is crucial for organism reproduction, development and likely stress responses. m6A mostly mediates its effect by recruiting reader proteins that either directly accommodate the modified residue in a hydrophobic pocket formed by their YTH domain, or otherwise have their affinity positively influenced by the presence of m6A. We firstly describe here the evolutionary history, and review known molecular and physiological roles of eukaryote YTH readers. In the second part, we present non YTH-proteins whose roles as m6A readers largely remain to be explored. The diversity and multiplicity of m6A readers together with the possibility to regulate their expression and function in response to various cues, offers a multitude of possible combinations to rapidly and finely tune gene expression patterns and hence cellular plasticity

The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5′TOP sequence

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5′TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5′ UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5′TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. A putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. These studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signaling to ribosome biogenesis

A bona fide La protein is required for embryogenesis in Arabidopsis thaliana

Searches in the Arabidopsis thaliana genome using the La motif as query revealed the presence of eight La or La-like proteins. Using structural and phylogenetic criteria, we identified two putative genuine La proteins (At32 and At79) and showed that both are expressed throughout plant development but at different levels and under different regulatory conditions. At32, but not At79, restores Saccharomyces cerevisiae La nuclear functions in non-coding RNAs biogenesis and is able to bind to plant 3′-UUU-OH RNAs. We conclude that these La nuclear functions are conserved in Arabidopsis and supported by At32, which we renamed as AtLa1. Consistently, AtLa1 is predominantly localized to the plant nucleoplasm and was also detected in the nucleolar cavity. The inactivation of AtLa1 in Arabidopsis leads to an embryonic-lethal phenotype with deficient embryos arrested at early globular stage of development. In addition, mutant embryonic cells display a nucleolar hypertrophy suggesting that AtLa1 is required for normal ribosome biogenesis. The identification of two distantly related proteins with all structural characteristics of genuine La proteins suggests that these factors evolved to a certain level of specialization in plants. This unprecedented situation provides a unique opportunity to dissect the very different aspects of this crucial cellular activity

LARP6 proteins in plants

International audienceRNA binding proteins, through control of mRNA fate and expression, are key players of organism development. The LARP family of RBPs sharing the La motif, are largely present in eukaryotes. They classify into five subfamilies which members acquired specific additional domains, including the RRM1 moiety which teams up with the La motif to form a versatile RNA binding unit. The LARP6 subfamily has had a peculiar history during plant evolution. While containing a single LARP6 in algae and non-vascular plants, they expanded and neofunctionalized into three subclusters in vascular plants. Studies from Arabidopsis thaliana, support that they acquired specific RNA binding properties and physiological roles. In particular LARP6C participates, through spatiotemporal control of translation, to male fertilization, a role seemingly conserved in maize. Interestingly, human LARP6 also acts in translation control and mRNA transport and similarly to LARP6C which is required for pollen tube guided elongation, is necessary to cell migration, through protrusion extension. This opens the possibility that some cellular and molecular functions of LARP6 were retained across eukaryote evolution. With their peculiar evolutionary history, plants provide a unique opportunity to uncover how La-module RNA binding properties evolved and identify species specific and basal roles of the LARP6 function. Deciphering of how LARP6, in particular LARP6C, acts at the molecular level, will foster novel knowledge on translation regulation and dynamics in changing cellular contexts. Considering the seemingly conserved function of LARP6C in male reproduction, it should fuel studies aimed at deriving crop species with improved seed yields

Immunity gate-keepers

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A comprehensive analysis of the La-motif protein superfamily

The extremely well-conserved La motif (LAM), in synergy with the immediately following RNA recognition motif (RRM), allows direct binding of the (genuine) La autoantigen to RNA polymerase III primary transcripts. This motif is not only found on La homologs, but also on La-related proteins (LARPs) of unrelated function. LARPs are widely found amongst eukaryotes and, although poorly characterized, appear to be RNA-binding proteins fulfilling crucial cellular functions. We searched the fully sequenced genomes of 83 eukaryotic species scattered along the tree of life for the presence of LAM-containing proteins. We observed that these proteins are absent from archaea and present in all eukaryotes (except protists from the Plasmodium genus), strongly suggesting that the LAM is an ancestral motif that emerged early after the archaea-eukarya radiation. A complete evolutionary and structural analysis of these proteins resulted in their classification into five families: the genuine La homologs and four LARP families. Unexpectedly, in each family a conserved domain representing either a classical RRM or an RRM-like motif immediately follows the LAM of most proteins. An evolutionary analysis of the LAM-RRM/RRM-L regions shows that these motifs co-evolved and should be used as a single entity to define the functional region of interaction of LARPs with their substrates. We also found two extremely well conserved motifs, named LSA and DM15, shared by LARP6 and LARP1 family members, respectively. We suggest that members of the same family are functional homologs and/or share a common molecular mode of action on different RNA baits

Fast and Efficient 5′P Degradome Library Preparation for Analysis of Co-Translational Decay in Arabidopsis

The recent development of high-throughput technologies based on RNA sequencing has allowed a better description of the role of post-transcriptional regulation in gene expression. In particular, the development of degradome approaches based on the capture of 5′monophosphate decay intermediates allows the discovery of a new decay pathway called co-translational mRNA decay. Thanks to these approaches, ribosome dynamics could now be revealed by analysis of 5′P reads accumulation. However, library preparation could be difficult to set-up for non-specialists. Here, we present a fast and efficient 5′P degradome library preparation for Arabidopsis samples. Our protocol was designed without commercial kit and gel purification and can be easily done in one working day. We demonstrated the robustness and the reproducibility of our protocol. Finally, we present the bioinformatic reads-outs necessary to assess library quality control

Synthesis and processing of tRNA-related SINE transcripts in Arabidopsis thaliana

Despite the ubiquitous distribution of tRNA-related short interspersed elements (SINEs) in eukaryotic species, very little is known about the synthesis and processing of their RNAs. In this work, we have characterized in detail the different RNA populations resulting from the expression of a tRNA-related SINE S1 founder copy in Arabidopsis thaliana. The main population is composed of poly(A)-ending (pa) SINE RNAs, while two minor populations correspond to full-length (fl) or poly(A) minus [small cytoplasmic (sc)] SINE RNAs. Part of the poly(A) minus RNAs is modified by 3′-terminal addition of C or CA nucleotides. All three RNA populations accumulate in the cytoplasm. Using a mutagenesis approach, we show that the poly(A) region and the 3′ end unique region, present at the founder locus, are both important for the maturation and the steady-state accumulation of the different S1 RNA populations. The observation that primary SINE transcripts can be post-transcriptionally processed in vivo into a poly(A)-ending species introduces the possibility that this paRNA is used as a retroposition intermediate