Isabelle Hatin

Impact of the six nucleotides downstream of the stop codon on translation termination

The efficiency of translation termination is influenced by local contexts surrounding stop codons. In Saccharomyces cerevisiae, upstream and downstream sequences act synergistically to influence the translation termination efficiency. By analysing derivatives of a leaky stop codon context, we initially demonstrated that at least six nucleotides after the stop codon are a key determinant of readthrough efficiency in S. cerevisiae. We then developed a combinatorial‐based strategy to identify poor 3′ termination contexts. By screening a degenerate oligonucleotide library, we identified a consensus sequence –CA(A/G)N(U/C/G)A–, which promotes >5% readthrough efficiency when located downstream of a UAG stop codon. Potential base pairing between this stimulatory motif and regions close to helix 18 and 44 of the 18S rRNA provides a model for the effect of the 3′ stop codon context on translation termination.

Nonsense-mediated decay mutants do not affect programmed -1 frameshifting.

Sequences in certain mRNAs program the ribosome to undergo a noncanonical translation event, translational frameshifting, translational hopping, or termination readthrough. These sequences are termed recoding sites, because they cause the ribosome to change temporarily its coding rules. Cis and trans-acting factors sensitively modulate the efficiency of recoding events. In an attempt to quantitate the effect of these factors we have developed a dual-reporter vector using the lacZ and luc genes to directly measure recoding efficiency. We were able to confirm the effect of several factors that modulate frameshift or readthrough efficiency at a variety of sites. Surprisingly, we were not able to confirm that the complex of factors termed the surveillance complex regulates translational frameshifting. This complex regulates degradation of nonsense codon-containing mRNAs and we confirm that it also affects the efficiency of nonsense suppression. Our data suggest that the surveillance complex is not a general regulator of translational accuracy, but that its role is closely tied to the translational termination and initiation processes.

Towards a computational model for −1 eukaryotic frameshifting sites

Abstract Motivation: Unconventional decoding events are now well acknowledged, but not yet well formalized. In this study, we present a bioinformatics analysis of eukaryotic −1 frameshifting, in order to model this event. Results: A consensus model has already been established for −1 frameshifting sites. Our purpose here is to provide new constraints which make the model more precise. We show how a machine learning approach can be used to refine the current model. We identify new properties that may be involved in frameshifting. Each of the properties found was experimentally validated. Initially, we identify features of the overall model that are to be simultaneously satisfied. We then focus on the following two components: the spacer and the slippery sequence. As a main result, we point out that the identity of the primary structure of the so-called spacer is of great importance. Availability: Sequences of the oligonucleotides in the functional tests are available at http://www.igmors.u-psud.fr/rousset/bioinformatics/ Contact: bekaert@igmors.u-psud.frjpforest@lri.frchris@lri.fr * To whom correspondence should be addressed.

Global translational impacts of the loss of the tRNA modification t6A in yeast

The universal tRNA modification t6A is found at position 37 of nearly all tRNAs decoding ANN codons. The absence of t6A37 leads to severe growth defects in baker’s yeast, phenotypes similar to those caused by defects in mcm5s2U34 synthesis. Mutants in mcm5s2U34 can be suppressed by overexpression of tRNALysUUU, but we show t6A phenotypes could not be suppressed by expressing any individual ANN decoding tRNA, and t6A and mcm5s2U are not determinants for each other’s formation. Our results suggest that t6A deficiency, like mcm5s2U deficiency, leads to protein folding defects, and show that the absence of t6A led to stress sensitivities (heat, ethanol, salt) and sensitivity to TOR pathway inhibitors. Additionally, L-homoserine suppressed the slow growth phenotype seen in t6A-deficient strains, and proteins aggregates and Advanced Glycation End-products (AGEs) were increased in the mutants. The global consequences on translation caused by t6A absence were examined by ribosome profiling. Interestingly, the absence of t6A did not lead to global translation defects, but did increase translation initiation at upstream non-AUG codons and increased frame-shifting in specific genes. Analysis of codon occupancy rates suggests that one of the major roles of t6A is to homogenize the process of elongation by slowing the elongation rate at codons decoded by high abundance tRNAs and I34:C3 pairs while increasing the elongation rate of rare tRNAs and G34:U3 pairs. This work reveals that the consequences of t6A absence are complex and multilayered and has set the stage to elucidate the molecular basis of the observed phenotypes.

Up-regulation of tRNA biosynthesis affects translational readthrough in maf1-Δ mutant of Saccharomyces cerevisiae

Abstract. Maf1p is a negative effector of RNA polymerase III in yeast. The maf1-Δ mutation caused an increase in the level of cellular tRNAs, but a decrease of translational readthrough at nonsense codons. Using the lacZ-luc dual gene reporter system, we detected an almost twofold diminution of UAA and UAG readthrough in maf1-Δ compared with the parental strain. The maf1-Δ mutation did not affect the rate of protein biosynthesis and growth at standard conditions, but resulted in temperature-sensitive growth on non-fermentable carbon sources. We examined the correlation of the temperature sensitive and antisuppression phenotypes of maf1-Δ using a colour phenotype assay in the ade2-1 SUP11 strain. Antisuppression, but not the temperature-sensitive growth defect, was compensated either by increased dosage of SUP11 or by [PSI+], the prion form of the translation termination factor Sup35p. Summarizing, the elevated tRNA levels in maf1-Δ increase translational fidelity and, independently, affect growth under special conditions.

RiboTools: a Galaxy toolbox for qualitative ribosome profiling analysis

MOTIVATION Ribosome profiling provides genome-wide information about translational regulation. However, there is currently no standard tool for the qualitative analysis of Ribo-seq data. We present here RiboTools, a Galaxy toolbox for the analysis of ribosome profiling (Ribo-seq) data. It can be used to detect translational ambiguities, stop codon readthrough events and codon occupancy. It provides a large number of plots for the visualisation of these events.

Molecular dissection of translation termination mechanism identifies two new critical regions in eRF1

Translation termination in eukaryotes is completed by two interacting factors eRF1 and eRF3. In Saccharomyces cerevisiae, these proteins are encoded by the genes SUP45 and SUP35, respectively. The eRF1 protein interacts directly with the stop codon at the ribosomal A-site, whereas eRF3—a GTPase protein—probably acts as a proofreading factor, coupling stop codon recognition to polypeptide chain release. We performed random PCR mutagenesis of SUP45 and screened the library for mutations resulting in increased eRF1 activity. These mutations led to the identification of two new pockets in domain 1 (P1 and P2) involved in the regulation of eRF1 activity. Furthermore, we identified novel mutations located in domains 2 and 3, which confer stop codon specificity to eRF1. Our findings are consistent with the model of a closed-active conformation of eRF1 and shed light on two new functional regions of the protein.

The case against the involvement of the NMD proteins in programmed frameshifting.

The complexity of the nonsense-mediated decay (NMD) system makes it difficult to study by comparing the expression of various single reporter constructs. The known effects of the NMD genes include a reduction both in mRNA stability (reviewed by Czaplinski et al., 1999) and in the efficiency of translational initiation (Muhlrad & Parker, 1999) of nonsense-containing plasmids as well as an apparent increase in the efficiency of translational termination as evidenced by increased readthrough of nonsense mutations (Bidou et al., 2000; Maderazo et al., 2000). The single reporter system can not distinguish among these effects and inference is required to determine which mechanism underlies any observed phenotypic effect on gene expression. It is particularly problematic to differentiate the effects of translation initiation accuracy from putative effects on translational frameshifting. The dual reporter system used in our work isolates the effect of translational frameshifting from effects on mRNA stability, initiation or termination. Much is made by Dinman et al. of the relative effects of various mutations, yet it remains unclear whether these are fundamental differences or simply differences in phenotypic strength of the various mutations.

Translation Analysis at the Genome Scale by Ribosome Profiling

Ribosome profiling is an emerging approach using deep sequencing of the mRNA part protected by the ribosome to study protein synthesis at the genome scale. This approach provides new insights into gene regulation at the translational level. In this review we describe the protocol to prepare polysomes and extract ribosome protected fragments before to deep sequence them.

Fibrillarin is essential for S-phase progression and neuronal differentiation in zebrafish dorsal midbrain and retina

Fibrillarin (Fbl) is a highly conserved protein that plays an essential role in ribosome biogenesis and more particularly in the methylation of ribosomal RNAs and rDNA histones. In cellular models, FBL was shown to play an important role in tumorigenesis and stem cell differentiation. We used the zebrafish as an in vivo model to study Fbl function during embryonic development. We show here that the optic tectum and the eye are severely affected by Fbl depletion whereas ventral regions of the brain are less impacted. The morphogenesis defects are associated with impaired neural differentiation and massive apoptosis. Polysome gradient experiments show that fbl mutant larvae display defects in ribosome biogenesis and activity. Strikingly, flow cytometry analyses revealed different S-phase profiles between wild-type and mutant cells, suggesting a defect in S-phase progression.