Biology subjects

Lessen, L. N.

Publications and source records attributed to Lessen, L. N..

4 recordsLinked to original sources

bioRxiv2021

Distinct ribosome states trigger diverse mRNA quality control pathways

Key protein adapters couple translation to mRNA decay on specific classes of problematic mRNAs in eukaryotes. Slow decoding on non-optimal codons leads to codon-optimality-mediated decay (COMD) and prolonged arrest at stall sites leads to no-go decay (NGD). The identities of the decay factors underlying these processes and the mechanisms by which they respond to translational distress remain open areas of investigation. We use carefully-designed reporter mRNAs to perform genetic screens and functional assays in S. cerevisiae. We characterize the roles of Hel2 and Syh1 in coordinating translational repression and mRNA decay on NGD reporter mRNAs, finding that Syh1 acts as the primary link to mRNA decay in NGD. Importantly, we observe that these NGD factors are not involved in the degradation of mRNAs enriched in non-optimal codons. Further, we establish that a key factor previously implicated in COMD, Not5, contributes modestly to the degradation of an NGD-targeted mRNA. Finally, we use ribosome profiling to reveal distinct ribosomal states associated with each reporter mRNA that readily rationalize the contributions of NGD and COMD factors to degradation of these reporters. Taken together, these results provide new mechanistic insight into the role of Syh1 in NGD and define the molecular triggers that determine how distinct pathways target mRNAs for degradation in yeast.

molecular biology↗

bioRxiv2021

Genetic screens identify connections between ribosome recycling and nonsense mediated decay

The decay of messenger RNA with a premature termination codon (PTC) by nonsense mediated decay (NMD) is an important regulatory pathway for eukaryotes and an essential pathway in mammals. NMD is typically triggered by the ribosome terminating at a stop codon that is aberrantly distant from the poly-A tail. Here, we use a fluorescence screen to identify factors involved in NMD in S. cerevisiae. In addition to the known NMD factors, including the entire UPF family (UPF1, UPF2 and UPF3), as well as NMD4 and EBS1, we identify factors known to function in post-termination recycling and characterize their contribution to NMD. We then use a series of modified reporter constructs that block both elongating and scanning ribosomes downstream of stop codons and demonstrate that a deficiency in recycling of 80S ribosomes or 40S subunits stabilizes NMD substrates. These observations in S. cerevisiae expand on recently reported data in mammals indicating that the 60S recycling factor ABCE1 is important for NMD (1, 2) by showing that increased activities of both elongating and scanning ribosomes (80S or 40S) in the 3UTR correlate with a loss of NMD. Author SummaryIn this work, we aim to understand the mechanism of targeting mRNAs for decay via the long-studied nonsense mediated decay (NMD) pathway. We demonstrate that efficient large and small subunit ribosome recycling are necessary components of NMD. We go on to provide evidence that either scanning or actively translating ribosomes in the 3UTR disrupt the decay of NMD targets. Our work highlights the importance of the composition of the 3UTR in NMD signaling and emphasizes the need for this region to indeed be untranslated for NMD to occur. Exon junction complexes (EJCs) in the 3UTR are known to induce NMD, however, in the budding yeast system used here, the NMD targets are EJC-free. Therefore, our data support a model in which factors other than EJCs may accumulate in the 3UTR and provide a signal for NMD.

molecular biology↗

bioRxiv2021

Mechanisms that ensure speed and fidelity in eukaryotic translation termination

Translation termination, which liberates a nascent polypeptide from the ribosome specifically at stop codons, must occur accurately and rapidly. We established single-molecule fluorescence assays to track the dynamics of ribosomes and two requisite release factors (eRF1 and eRF3) throughout termination using an in vitro-reconstituted yeast translation system. We found that the two eukaryotic release factors bind together to recognize stop codons rapidly and elicit termination via a tightly regulated, multi-step process that resembles tRNA selection during translation elongation. Because the release factors are conserved from yeast to humans, the molecular events that underlie yeast translation termination are likely broadly fundamental to eukaryotic protein synthesis. One Sentence SummaryDirect visualization of eukaryotic translation reveals the dynamics of termination at stop codons.

biochemistry↗

bioRxiv2019

Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts

Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination of in vitro biochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an in vitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.

biochemistry↗