Molecular mechanism of the ribosome and functions of translational regulation

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

We have a longstanding interest in the processes of translation termination and ribosome recycling, the final steps in the translation of the genetic code. We also study how ribosomes that become stalled while translating are detected by quality control machinery in the cell and activate signaling pathways. Our goal is to uncover the molecular mechanism for how these processes occur and understand how they are regulated to carry out biological functions and maintain human health. The termination step of translation is critical for releasing the completed protein from the ribosome so that it can carry out functions in the cell. The termination process begins when the ribosome reaches a stop codon and the release factors (eRF1/3) are recruited to the ribosome to catalyze this process. Termination is generally efficient and it is only in rare cases that the ribosome reads through the stop codon by incorporating a tRNA with an anticodon that nearly pairs with the stop codon. The consequence of a readthrough event is that the ribosome continues translating downstream, adding a C-terminal extension to the protein that may alter its function. Many genes permit readthrough to occur at relatively high (10%) frequency. Our goal is to uncover the mechanism for how these programmed readthrough events occur, how they are regulated, and how C-terminal protein extensions affect function. We developed assays to directly observe readthrough events in the cell using fluorescence microscopy and reporter genes that encode protein arrays that become fluorescent as they emerge from the ribosome, thus allowing translation of single mRNAs to be imaged (suntag or moontag). In addition, we have developed computational tools for tracking single molecules in images and assessing their fluorescence intensity over time. We are currently measuring differences in readthrough kinetics (stochastic vs burst-like) for programmed readthrough events. We find that burst events are correlated with the number of ribosomes loaded onto a particular transcript. This suggests that interactions between ribosomes, potentially at the stop codon, could influence the efficiency of termination. Since ribosome queuing can be modulated by stress or other factors, such as developmental state or localization in the cell, readthrough may also be modulated and changes in readthrough efficiency could help the cell respond to stress. We are also studying a related problem of premature translation termination. In premature termination, the ribosome stops translating at a stop codon that lies upstream of the canonical stop codon. Ribosomes that undergo premature termination can be sensed by a pathway called nonsense-mediated decay (NMD) that leads to degradation of the mRNA and is important for eliminating aberrant mRNAs that do not encode full-length proteins. However, the NMD pathway is known to target many apparently normal transcripts and is therefore thought to play additional roles in gene regulation. We hypothesized that cryptic translation events could lead to premature termination so we developed a 40S ribosome profiling technique that allows us to identify stop codons that are actively used for premature translation termination. Using this, we found many cryptic translation events that lead to termination that is in 5UTRs or within out-of-frame regions of coding sequences. In 5UTRs, we also found evidence for premature translation termination on long undecoded transcript isoforms (LUTIs), an important class of RNAs that are thought to be generated to carry out regulatory roles in the cell. In particular, promoters for LUTIs tend to be far upstream of canonical promoters and often include many upstream open reading frames (uORFs). Premature termination after translation of these uORFs results in NMD, suggesting that NMD is important for ensuring these transcripts are degraded once they emerge from the nucleus. Using this approach of 40S ribosome profiling to find novel NMD targets, we can account for the vast majority of NMD cases in the cell with cryptic translation in 5UTRs or short out-of-frame ORFs withing coding sequences. We are also interested in understanding how the cell responds to ribosomes that stall while translating. Such events have to be resolved to prevent upstream ribosomes from accumulating on the mRNA behind the arrested ribosome. In our ongoing work with Gustavo Silva's lab (Duke University), we have examined mechanisms by which cells detect these queues of collided ribosomes, particularly under stress conditions, by using a modified ribosome profiling approach to detect long footprints created by disomes (ribosomes that have bumped into each other). We have applied this technique to examine the role of ribosome stalling during oxidative stress. We found this stress causes ribosomes to become arrested on isoleucine-proline codon pairs, in particular, and that the stalling is dependent on the ubiquitin conjugase Rad6. Rad6 is known to facilitate the conjugation of K63 ubiquitin chains to many ribosomal proteins. Loss of this activity appears to enable the ribosome to more rapidly translate under oxidative stress without stalling at Ile-Pro motifs. These results therefore offer an explanation for the underlying function of these ubiquitin chains. In addition, we worked collaboratively with the lab of Voula Mili (NCI/NIH) to show that the slowed speed of elongation, and in particular formation of disomes, can influence the localization of the nascent peptide when it is released the ribosome. We found that for the gene NET1, slow translation directs the protein product of the gene to the nucleus while faster translation favors localization to the cell periphery. Our work has also examined how translation changes during viral infection and promotes the innate immune response. In particular, we have examined the role of RNase L, an endonuclease that is activated when viral RNA is detected in the cell. RNase L activation is known to cause widespread mRNA decay and leads to eventual apoptosis and elimination of the infected cell. However, it is unclear whether the degraded transcriptome is still translated and whether it serves any function. To address this, we used ribosome profiling on RNase L activated cells to examine how the residual transcriptome is translated. Our initial results showed that mRNA degradation intermediate fragments in the cell can be translated, leading to broad translation of all parts of a mRNA since initiation of translation occurs all along the mRNAs length. More recently, we found that activation of RNase L also leads to the activation of the ZAK-alpha kinase, a sensor of disomes, and its downstream transcriptional program that eventually leads to apoptosis. Our data suggest that translation of mRNA fragments could promote disome formation whenever ribosomes reach the 3 end of a mRNA fragment and arrest, allowing upstream ribosomes to collide with them. To test this hypothesis, we transfected a different endonuclease, RNase A, into cells to see if similar phenotypes emerged as when RNase L was active. Our data similarly show activation of ZAK-alpha and translation of mRNA fragments. These results therefore reveal how degradation of RNA globally in the cell can activate a pro-apoptotic transcriptional program to help clear viral infections.