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 study the mechanism by which the ribosome translates the genetic code and how translation is regulated to control gene expression in response to changing environmental conditions, such as nutrient limitation, oxidative stress, and viral infection. In particular, we aim to understand the process of translation termination and ribosome recycling that ends translation and removes ribosomes from mRNAs. We also study the mechanisms that detect ribosomes that become stalled while translating. We broadly expect that discoveries from this research will offer new targets for therapies against aging, cancer, and infectious disease. We utilize a combined experimental approach, employing ribosome profiling and RNA-Seq, biochemistry, and single-molecule fluorescence microscopy in live cells. 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 the stop codon and a heterodimer of release factors (eRF1/3) is recruited to catalyze this process. Termination is generally efficient compared to the rare process of readthrough, where an amino acid is misincorporated instead. After a readthrough event, the ribosome continues translating downstream of the stop codon and creates a C-terminal extension on the protein. However, 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-terminally extended proteins affect function. We have developed assays to directly observe readthrough events in the cell using fluorescence microscopy and reporter genes that encode protein arrays that become fluorescent during translation (suntag or moontag). Using these, we are now characterizing differences in readthrough kinetics (stochastic vs burst-like) for programmed readthrough events. We find that burst events are correlated with queues of ribosomes, suggesting that ribosome interactions can affect termination efficiency. Since ribosome queuing can be modulated by stress or potentially other factors, such as developmental state or localization in the cell, readthrough may be modulated to help the cell respond to stress. We are also studying cases of premature translation termination, where the ribsosome reaches a stop codon and ends translation before reaching the 3' end of the gene. Such events trigger 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. To search for cryptic translation events that would cause premature translation termination in normal cells, we employed the 40S ribosome profiling technique we developed to identify stop codons that were being actively used for premature translation termination. One class of stop codons we found includes those internal to coding sequences, indicative of a process called leaky scanning. Leaky scanning occurs when the 40S ribosome binds to an mRNA but fails to find the main AUG start codon. Instead, it initiates translation downstream at a short (out of frame) ORF that results in a premature termination event. We also found evidence for premature translation termination on long undecoded transcript isoforms (LUTIs), a key class of RNA that are thought to be generated as a consequence of transcription events that generate mRNAs that are primarily regulatory in nature. In particular, far upstream promoters are used to make LUTIs and they include 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 silenced and do not produce a protein product. We are also interested in understanding how the cell monitors translating ribosomes for aberrant events where the ribosome stalls during its translation cycle. Such events have to be resolved to prevent ribosomes from accumulating on the mRNA behind the arrested ribosome. In our ongoing work with Gustavo Silva's lab, we have examined mechanisms by which cells detect these queues of collided ribosomes (disomes), particularly under stress conditions, by using a modified ribosome profiling approach to detect footprints created by disomes. We have applied this technique to examine the role of ribosome stalling during oxidative stress, a condition that causes ribosomes to become arrested on isoleucine-proline codon pairs. We found these stalling events are 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. In addition, we see evidence that the ubiquitins affect initiation, by limiting the availability of some initiation factors. These results offer an important insight on the underlying function of these ubiquitin chains. We are interested in in further understanding how these functions promote resistance to oxidative stress. 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 potentially facilitates this process, or under low-level activation, triggers alternative mechanisms to clear the virus without causing apoptosis. To address this, we used ribosome profiling on RNase L activated cells to examine how the residual transcriptome is translated. Strikingly, we observed increased translation in non-coding regions, including 5' and 3' untranslated regions and out-of-frame ORFs in coding sequences. Moreover, the effects depended on the cleavage activity of RNase L, suggesting a model where ribosomes initiate translation on mRNA decay fragments and translate any ORF that is encountered. Consistent with this, we found mRNA decay fragments were detectable in the cell. This work establishes a novel mechanism for promoting translation of alternative open reading frames during viral infection. We are now pursuing two additional directions. First, we are examining how activation of other RNases or inhibition of decay pathways increases the number of mRNA fragments in the cell. We have found that fragments made available under these conditions can also be translated, suggesting that the model where degradation of mRNA by RNase L promotes translation of alternative ORFs is also used by other endonucleases, such IRE1 under stress. Second, we are exploring how global loss of mRNA triggers and modulates other pathways that are key to enhancing the cell's response to the virus. In particular, we have found evidence for how loss of overall gene expression is coupled into specific transcriptional events that trigger important stress-response pathways.