Gwenael Badis

Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic surveillance pathway known to degrade mRNAs containing premature termination codons (PTCs). mRNA recognition by the Upf1, Upf2 and Upf3 NMD factors is favoured by long distances between the stop codon and the poly(A)-binding protein (Pab1) binding site. Using Nanopore direct RNA sequencing, we now show that PTC-containing NMD targets account for only 6% of Upf1-associated RNA and have long poly(A) tails, indicating that Upf1-binding occurs prior to their deadenylation. Conversely, most Upf1-associated mRNAs have short poly(A) tails, lack a PTC and correspond to highly expressed genes. A short poly(A) tail is thus an important feature of a new class of Upf1 targets, redefining the scope of the NMD RNA degradation pathway. Recognition of short poly(A)-tailed mRNAs by Upf1, Upf2 and Upf3 (the NMD machinery) triggers their decapping, uncovering a hitherto unknown role of the NMD machinery in the degradation of these transcripts.

Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic surveillance pathway known to degrade mRNAs containing premature termination codons (PTCs). A distance long enough between the stop codon and the poly(A)-binding protein (Pab1) is required for mRNA recognition by the NMD factors Upf1, Upf2 and Upf3. Using Nanopore direct RNA sequencing, we show that PTC-containing NMD targets account for only 6% of Upf1-associated RNA and have long poly(A) tails, indicating that Upf1-binding occurs prior to RNA deadenylation. Conversely, most Upf1-associated mRNAs have short poly(A) tails, lack a PTC and correspond to highly expressed genes. A short poly(A) tail is thus an important feature of NMD targets, redefining the scope of this RNA degradation pathway. We propose a model in which loss of Pab1-binding to short poly(A)-tailed mRNAs impairs translation termination and dictates the recruitment of the NMD machinery, uncovering a hitherto unknown role of NMD in the degradation of these transcripts.

SUMMARY: Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic surveillance pathway known to degrade mRNAs containing premature termination codons (PTCs). A distance long enough between the stop codon and the poly(A)-binding protein (Pab1) is required for mRNA recognition by the NMD factors Upf1, Upf2 and Upf3. Using Nanopore direct RNA sequencing, we show that PTC-containing NMD targets account for only 6% of Upf1-associated RNA and have long poly(A) tails, indicating that Upf1-binding occurs prior to RNA deadenylation. Conversely, most Upf1-associated mRNAs have short poly(A) tails, lack a PTC and correspond to highly expressed genes. A short poly(A) tail is thus an important feature of NMD targets, redefining the scope of this RNA degradation pathway. We propose a model in which loss of Pab1-binding to short poly(A)-tailed mRNAs impairs translation termination and dictates the recruitment of the NMD machinery, uncovering a hitherto unknown role of NMD in the degradation of these transcripts.

RNA stability, important for eukaryotic gene expression, is thought to depend on deadenylation rates, with shortened poly(A) tails triggering decapping and 5′ to 3′ degradation. In contrast to this view, recent large-scale studies indicate that the most unstable mRNAs have, on average, long poly(A) tails. To clarify the role of deadenylation in mRNA decay, we first modeled mRNA poly(A) tail kinetics and mRNA stability in yeast. Independent of deadenylation rates, differences in mRNA decapping rates alone were sufficient to explain current large-scale results. To test the hypothesis that deadenylation and decapping are uncoupled, we used rapid depletion of decapping and deadenylation enzymes and measured changes in mRNA levels, poly(A) length and stability, both transcriptome-wide and with individual reporters. These experiments revealed that perturbations in poly(A) tail length did not correlate with variations in mRNA stability. Thus, while deadenylation may be critical for specific regulatory mechanisms, our results suggest that for most yeast mRNAs, it is not critical for mRNA decapping and degradation.

27 years after the yeast genome sequencing, the function of many ORFs remain unknown. Despite the evolutionary distance between human and yeast, homology with the conserved DEAH/DExH-box helicase domains allowed us to list DHX29, DHX36 and DHX57 as three putative homologs of the yeast Ylr419wp. Functional studies first linked the Ylr419w protein to the translating ribosome and cross-linking and analysis of cDNA (CRAC) experiments determined the precise region of Ylr419wp in contact with the ribosome. It corresponds to the loop of the h16 helix in the 18S rRNA designing the translation initiation factor DHX29, as the functional homolog of Ylr419wp.

Gene expression and its regulation depend on mRNA degradation. In eukaryotes, degradation is controlled by deadenylation rates, since a short poly(A) tail is considered to be the signal that activates decapping and triggers mRNA degradation. In contrast to this view, we show that global stability of mRNAs can be explained by variations in decapping speed alone. Rapid decapping of unstable mRNAs, for example, allows little time for deadenylation, which explains their longer than average poly(A) tails. As predicted by modeling of RNA degradation kinetics, mRNA stabilization in the absence of decapping led to a decrease in the length of the poly(A) tail, while depletion of deadenylases only increased the tail length. Our results suggest that decapping activation dictates mRNA stability independent of the deadenylation speed. One-Sentence Summary Unstable mRNAs are characterized by rapid 5’ cap removal, independent of a prior shortening of the poly(A) tail.

Pervasive transcription generates many unstable non-coding transcripts in budding yeast. The transcription of such noncoding RNAs, in particular antisense RNAs (asRNAs), has been shown in a few examples to repress the expression of the associated mRNAs. Yet, such mechanism is not known to commonly contribute to the regulation of a given class of genes. Using a mutant context that stabilized pervasive transcripts, we observed that the least expressed mRNAs during the exponential phase were associated with high levels of asRNAs. These asRNAs also overlapped their corresponding gene promoters with a much higher frequency than average. Interrupting antisense transcription of a subset of genes corresponding to quiescence-enriched mRNAs restored their expression. The underlying mechanism acts in cis and involves several chromatin modifiers. Our results convey that transcription interference represses up to 30% of the 590 least expressed genes, which includes 163 genes with quiescence-enriched mRNAs. We also found that pervasive transcripts constitute a higher fraction of the transcriptome in quiescence relative to the exponential phase, consistent with gene expression itself playing an important role to suppress pervasive transcription. Accordingly, the HIS1 asRNA, normally only present in quiescence, is expressed in exponential phase upon HIS1 mRNA transcription interruption.

Pervasive transcription generates many unstable non-coding transcripts in budding yeast. The transcription of such noncoding RNAs, in particular antisense RNAs (asRNAs), has been shown in a few examples to repress the expression of the associated mRNAs. Yet, such mechanism is not known to commonly contribute to the regulation of a given class of genes. Using a mutant context that stabilised pervasive transcripts, we observed that the least expressed mRNAs during the exponential phase were associated with high levels of asRNAs. These asRNAs also overlapped their corresponding gene promoters with a much higher frequency than average. Interrupting antisense transcription of a subset of genes corresponding to quiescence-enriched mRNAs restored their expression. The underlying mechanism acts in cis and involves several chromatin modifiers. Our results convey that transcription interference represses up to 30% of the 590 least expressed genes, which includes 163 genes with quiescence-enriched mRNAs. We also found that pervasive transcripts constitute a higher fraction of the transcriptome in quiescence relative to the exponential phase, consistent with gene expression itself playing an important role to suppress pervasive transcription. Accordingly, the HIS1 asRNA, normally only present in quiescence, is expressed in exponential phase upon HIS1 mRNA transcription interruption.

Understanding how genes are regulated, decoding their “regulome”, is one of the main challenges of the post-genomic era. Here, we describe the in vitro method we used to associate cis-regulatory sites with cognate trans-regulators by characterizing the DNA-binding specificity of the vast majority of yeast transcription factors using Protein Binding Microarrays. This approach can be implemented to any given organism.

Ribosome biogenesis requires >300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24, suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing sites C1 and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7(L)b within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation.