Endonucleolytic RNA cleavage drives changes in gene expression during the innate immune response.

Viral infection triggers several double-stranded RNA (dsRNA) sensors that lead to changes in gene expression in the cell. One of these sensors activates an endonuclease, ribonuclease L (RNase L), that cleaves single-stranded RNA. However, how the resultant widespread RNA fragmentation affects gene expression is not fully understood. Here, we show that this fragmentation induces the ribotoxic stress response via ZAKα, potentially through stalled ribosomes and/or ribosome collisions. The p38 and JNK pathways that are activated as part of this response promote outcomes that inhibit the virus, such as programmed cell death. We also show that RNase L limits the translation of stress-responsive genes. Intriguingly, we found that the activity of the generic endonuclease, RNase A, recapitulates many of the same molecular phenotypes as activated RNase L, demonstrating how widespread RNA cleavage can evoke an antiviral program.

The ubiquitin conjugase Rad6 mediates ribosome pausing during oxidative stress.

Oxidative stress causes K63-linked ubiquitination of ribosomes by the E2 ubiquitin conjugase Rad6. How Rad6-mediated ubiquitination of ribosomes affects translation, however, is unclear. We therefore perform Ribo-seq and Disome-seq in Saccharomyces cerevisiae and show that oxidative stress causes ribosome pausing at specific amino acid motifs, which also leads to ribosome collisions. However, these redox-pausing signatures are lost in the absence of Rad6 and do not depend on the ribosome-associated quality control (RQC) pathway. We also show that Rad6 is needed to inhibit overall translation in response to oxidative stress and that its deletion leads to increased expression of antioxidant genes. Finally, we observe that the lack of Rad6 leads to changes during translation that affect activation of the integrated stress response (ISR) pathway. Our results provide a high-resolution picture of the gene expression changes during oxidative stress and unravel an additional stress response pathway affecting translation elongation.

Endonucleolytic RNA cleavage drives changes in gene expression during the innate immune response.

Viral infection triggers several dsRNA sensors that lead to changes in gene expression in the cell. One of these sensors activates an endonuclease, RNase L, that cleaves single stranded RNA. However, how the resultant widespread RNA fragmentation affects gene expression is not fully understood. Here we show that this fragmentation induces the Ribotoxic Stress Response via ZAKα, potentially through ribosome collisions. The p38 and JNK pathways that are activated as part of this response promote outcomes that inhibit the virus, such as programmed cell death. We also show that RNase L limits the translation of stress-responsive genes, including antiviral IFIT mRNAs and GADD34 that encodes an antagonist of the Integrated Stress Response. Intriguingly, we found the activity of the generic endonuclease, RNase A, recapitulates many of the same molecular phenotypes as activated RNase L, demonstrating how widespread RNA cleavage can evoke an antiviral program.

Is there a localized role for translational quality control?

It is known that mRNAs and the machinery that translates them are not uniformly distributed throughout the cytoplasm. As a result, the expression of some genes is localized to particular parts of the cell and this makes it possible to carry out important activities, such as growth and signaling, in three-dimensional space. However, the functions of localized gene expression are not fully understood, and the underlying mechanisms that enable localized expression have not been determined in many cases. One consideration that could help in addressing these challenges is the role of quality control (QC) mechanisms that monitor translating ribosomes. On a global level, QC pathways are critical for detecting aberrant translation events, such as a ribosome that stalls while translating, and responding by activating stress pathways and resolving problematic ribosomes and mRNAs at the molecular level. However, it is unclear how these pathways, even when uniformly active throughout the cell, affect local translation. Importantly, some QC pathways have themselves been reported to be enriched in the proximity of particular organelles, but the extent of such localized activity remains largely unknown. Here, we describe the major QC pathways and review studies that have begun to explore their roles in localized translation. Given the limited data in this area, we also pose broad questions about the possibilities and limitations for how QC pathways could facilitate localized gene expression in the cell with the goal of offering ideas for future experimentation.

mRNA location and translation rate determine protein targeting to dual destinations.

Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that mRNA location and translation rate can also determine protein targeting by modulating protein binding to specific interacting partners. Peripheral localization of the NET1 mRNA and fast translation lead to higher cytosolic retention of the NET1 protein by promoting its binding to the membrane-associated scaffold protein CASK. By contrast, perinuclear mRNA location and/or slower translation rate favor nuclear targeting by promoting binding to importins. This mRNA location-dependent mechanism is modulated by physiological stimuli and profoundly impacts NET1 function in cell motility. These results reveal that the location of protein synthesis and the rate of translation elongation act in coordination as a "partner-selection" mechanism that robustly influences protein distribution and function.

mRNA Location and Translation Rate Determine Protein Targeting to Dual Destinations.

Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that protein targeting can additionally be determined by mRNA location and translation rate, through modulating protein binding to specific interacting partners. Peripheral localization of the NET1 mRNA and fast translation lead to higher cytosolic retention of the NET1 protein, through promoting its binding to the membrane-associated scaffold protein CASK. By contrast, perinuclear mRNA location and/or slower translation rate favor nuclear targeting, through promoting binding to importins. This mRNA location-dependent mechanism is modulated by physiological stimuli and profoundly impacts NET1 function in cell motility. These results reveal that the location of protein synthesis and the rate of translation elongation act in coordination as a 'partner-selection' mechanism that robustly influences protein distribution and function.

Rebirth of the translational machinery: The importance of recycling ribosomes.

Translation of the genetic code occurs in a cycle where ribosomes engage mRNAs, synthesize protein, and then disengage in order to repeat the process again. The final part of this process-ribosome recycling, where ribosomes dissociate from mRNAs-involves a complex molecular choreography of specific protein factors to remove the large and small subunits of the ribosome in a coordinated fashion. Errors in this process can lead to the accumulation of ribosomes at stop codons or translation of downstream open reading frames (ORFs). Ribosome recycling is also critical when a ribosome stalls during the elongation phase of translation and must be rescued to allow continued translation of the mRNA. Here we discuss the molecular interactions that drive ribosome recycling, and their regulation in the cell. We also examine the consequences of inefficient recycling with regards to disease, and its functional roles in synthesis of novel peptides, regulation of gene expression, and control of mRNA-associated proteins. Alterations in ribosome recycling efficiency have the potential to impact many cellular functions but additional work is needed to understand how this regulatory power is utilized.

40S ribosome profiling reveals distinct roles for Tma20/Tma22 (MCT-1/DENR) and Tma64 (eIF2D) in 40S subunit recycling.

The recycling of ribosomes at stop codons for use in further rounds of translation is critical for efficient protein synthesis. Removal of the 60S subunit is catalyzed by the ATPase Rli1 (ABCE1) while removal of the 40S is thought to require Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR). However, it remains unclear how these Tma proteins cause 40S removal and control reinitiation of downstream translation. Here we used a 40S ribosome footprinting strategy to directly observe intermediate steps of ribosome recycling in cells. Deletion of the genes encoding these Tma proteins resulted in broad accumulation of unrecycled 40S subunits at stop codons, directly establishing their role in 40S recycling. Furthermore, the Tma20/Tma22 heterodimer was responsible for a majority of 40S recycling events while Tma64 played a minor role. Introduction of an autism-associated mutation into TMA22 resulted in a loss of 40S recycling activity, linking ribosome recycling and neurological disease.

Context-specific action of macrolide antibiotics on the eukaryotic ribosome.

Macrolide antibiotics bind in the nascent peptide exit tunnel of the bacterial ribosome and prevent polymerization of specific amino acid sequences, selectively inhibiting translation of a subset of proteins. Because preventing translation of individual proteins could be beneficial for the treatment of human diseases, we asked whether macrolides, if bound to the eukaryotic ribosome, would retain their context- and protein-specific action. By introducing a single mutation in rRNA, we rendered yeast Saccharomyces cerevisiae cells sensitive to macrolides. Cryo-EM structural analysis showed that the macrolide telithromycin binds in the tunnel of the engineered eukaryotic ribosome. Genome-wide analysis of cellular translation and biochemical studies demonstrated that the drug inhibits eukaryotic translation by preferentially stalling ribosomes at distinct sequence motifs. Context-specific action markedly depends on the macrolide structure. Eliminating macrolide-arrest motifs from a protein renders its translation macrolide-tolerant. Our data illuminate the prospects of adapting macrolides for protein-selective translation inhibition in eukaryotic cells.

Activation of the antiviral factor RNase L triggers translation of non-coding mRNA sequences.

Ribonuclease L (RNase L) is activated as part of the innate immune response and plays an important role in the clearance of viral infections. When activated, it endonucleolytically cleaves both viral and host RNAs, leading to a global reduction in protein synthesis. However, it remains unknown how widespread RNA decay, and consequent changes in the translatome, promote the elimination of viruses. To study how this altered transcriptome is translated, we assayed the global distribution of ribosomes in RNase L activated human cells with ribosome profiling. We found that RNase L activation leads to a substantial increase in the fraction of translating ribosomes in ORFs internal to coding sequences (iORFs) and ORFs within 5' and 3' UTRs (uORFs and dORFs). Translation of these alternative ORFs was dependent on RNase L's cleavage activity, suggesting that mRNA decay fragments are translated to produce short peptides that may be important for antiviral activity.

A cellular handbook for collided ribosomes: surveillance pathways and collision types.

Translating ribosomes slow down or completely stall when they encounter obstacles on mRNAs. Such events can lead to ribosomes colliding with each other and forming complexes of two (disome), three (trisome) or more ribosomes. While these events can activate surveillance pathways, it has been unclear if collisions are common on endogenous mRNAs and whether they are usually detected by these cellular pathways. Recent genome-wide surveys of collisions revealed widespread distribution of disomes and trisomes across endogenous mRNAs in eukaryotic cells. Several studies further hinted that the recognition of collisions and response to them by multiple surveillance pathways depend on the context and duration of the ribosome stalling. This review considers recent efforts in the identification of endogenous ribosome collisions and cellular pathways dedicated to sense their severity. We further discuss the potential role of collided ribosomes in modulating co-translational events and contributing to cellular homeostasis.

Disome and Trisome Profiling Reveal Genome-wide Targets of Ribosome Quality Control.

The ribosome-associated protein quality control (RQC) system that resolves stalled translation events is activated when ribosomes collide and form disome, trisome, or higher-order complexes. However, it is unclear whether this system distinguishes collision complexes formed on defective mRNAs from those with functional roles on endogenous transcripts. Here, we performed disome and trisome footprint profiling in yeast and found collisions were enriched on diverse sequence motifs known to slow translation. When 60S recycling was inhibited, disomes accumulated at stop codons and could move into the 3' UTR to reinitiate translation. The ubiquitin ligase and RQC factor Hel2/ZNF598 generally recognized collisions but did not induce degradation of endogenous transcripts. However, loss of Hel2 triggered the integrated stress response, via phosphorylation of eIF2α, thus linking these pathways. Our results suggest that Hel2 has a role in sensing ribosome collisions on endogenous mRNAs, and such events may be important for cellular homeostasis.

Hcr1/eIF3j Is a 60S Ribosomal Subunit Recycling Accessory Factor In Vivo.

Hcr1/eIF3j is a sub-stoichiometric subunit of eukaryotic initiation factor 3 (eIF3) that can dissociate the post-termination 40S ribosomal subunit from mRNA in vitro. We examine this ribosome recycling role in vivo by ribosome profiling and reporter assays and find that loss of Hcr1 leads to reinitiation of translation in 3' UTRs, consistent with a defect in recycling. However, the defect appears to be in the recycling of the 60S subunit, rather than the 40S subunit, because reinitiation does not require an AUG codon and is suppressed by overexpression of the 60S dissociation factor Rli1/ABCE1. Consistent with a 60S recycling role, overexpression of Hcr1 cannot compensate for loss of 40S recycling factors Tma64/eIF2D and Tma20/MCT-1. Intriguingly, loss of Hcr1 triggers greater expression of RLI1 via an apparent feedback loop. These findings suggest Hcr1/eIF3j is recruited to ribosomes at stop codons and may coordinate the transition to a new round of translation.

Tma64/eIF2D, Tma20/MCT-1, and Tma22/DENR Recycle Post-termination 40S Subunits In Vivo.

The recycling of ribosomal subunits after translation termination is critical for efficient gene expression. Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR) function as 40S recycling factors in vitro, but it is unknown whether they perform this function in vivo. Ribosome profiling of tma deletion strains revealed 80S ribosomes queued behind the stop codon, consistent with a block in 40S recycling. We found that unrecycled ribosomes could reinitiate translation at AUG codons in the 3' UTR, as evidenced by peaks in the footprint data and 3' UTR reporter analysis. In vitro translation experiments using reporter mRNAs containing upstream open reading frames (uORFs) further established that reinitiation increased in the absence of these proteins. In some cases, 40S ribosomes appeared to rejoin with 60S subunits and undergo an 80S reinitiation process in 3' UTRs. These results support a crucial role for Tma64, Tma20, and Tma22 in recycling 40S ribosomal subunits at stop codons and translation reinitiation.

Regulated Ire1-dependent mRNA decay requires no-go mRNA degradation to maintain endoplasmic reticulum homeostasis in S. pombe.

The unfolded protein response (UPR) monitors and adjusts the protein folding capacity of the endoplasmic reticulum (ER). In S. pombe, the ER membrane-resident kinase/endoribonuclease Ire1 utilizes a mechanism of selective degradation of ER-bound mRNAs (RIDD) to maintain homeostasis. We used a genetic screen to identify factors critical to the Ire1-mediated UPR and found several proteins, Dom34, Hbs1 and Ski complex subunits, previously implicated in ribosome rescue and mRNA no-go-decay (NGD). Ribosome profiling in ER-stressed cells lacking these factors revealed that Ire1-mediated cleavage of ER-associated mRNAs results in ribosome stalling and mRNA degradation. Stalled ribosomes iteratively served as a ruler to template precise, regularly spaced upstream mRNA cleavage events. This clear signature uncovered hundreds of novel target mRNAs. Our results reveal that the UPR in S. pombe executes RIDD in an intricate interplay between Ire1, translation, and the NGD pathway, and establish a critical role for NGD in maintaining ER homeostasis.

IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts.

Here we investigated in primary human erythroid tissues a downstream element of the heterochronic let-7 miRNA pathway, the insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), for its potential to affect the hemoglobin profiles in human erythroblasts. Comparison of adult bone marrow to fetal liver lysates demonstrated developmental silencing in IGF2BP1. Erythroid-specific overexpression of IGF2BP1 caused a nearly complete and pancellular reversal of the adult pattern of hemoglobin expression toward a more fetal-like phenotype. The reprogramming of hemoglobin expression was achieved at the transcriptional level by increased gamma-globin combined with decreased beta-globin transcripts resulting in gamma-globin rising to 90% of total beta-like mRNA. Delta-globin mRNA was reduced to barely detectable levels. Alpha-globin levels were not significantly changed. Fetal hemoglobin achieved levels of 68.6 ± 3.9% in the IGF2BP1 overexpression samples compared with 5.0 ± 1.8% in donor matched transduction controls. In part, these changes were mediated by reduced protein expression of the transcription factor BCL11A. mRNA stability and polysome studies suggest IGF2BP1 mediates posttranscriptional loss of BCL11A. These results suggest a mechanism for chronoregulation of fetal and adult hemoglobin expression in humans.

Translation of poly(A) tails leads to precise mRNA cleavage.

Translation of poly(A) tails leads to mRNA cleavage but the mechanism and global pervasiveness of this "nonstop/no-go" decay process is not understood. Here we performed ribosome profiling (in a yeast strain lacking exosome function) of short 15-18 nucleotides mRNA footprints to identify ribosomes stalled at 3' ends of mRNA decay intermediates. In this background, we found mRNA cleavage extending hundreds of nucleotides upstream of ribosome stalling in poly(A) and predominantly in one reading frame. These observations suggest that decay-triggering endonucleolytic cleavage is closely associated with the ribosome. Surprisingly, ribosomes appeared to accumulate (i.e., stall) in the transcriptome when as few as three consecutive ORF-internal lysine codons were positioned in the A, P, and E sites though significant mRNA degradation was not observed. Endonucleolytic cleavage was found, however, at sites of premature polyadenylation (encoding polylysine) and rescue of the ribosomes stalled at these sites was dependent on Dom34. These results suggest this process may be critical when changes in the polyadenylation site occur during development, tumorigenesis, or when translation termination/recycling is impaired.

Silence without stress.

Two mechanisms ensure that the mRNA encoding Hac1 protein, a transcription factor involved in the unfolded protein response, is only translated when it is needed.

Rli1/ABCE1 Recycles Terminating Ribosomes and Controls Translation Reinitiation in 3'UTRs In Vivo.

To study the function of Rli1/ABCE1 in vivo, we used ribosome profiling and biochemistry to characterize its contribution to ribosome recycling. When Rli1 levels were diminished, 80S ribosomes accumulated both at stop codons and in the adjoining 3'UTRs of most mRNAs. Frequently, these ribosomes reinitiated translation without the need for a canonical start codon, as small peptide products predicted by 3'UTR ribosome occupancy in all three reading frames were confirmed by western analysis and mass spectrometry. Eliminating the ribosome-rescue factor Dom34 dramatically increased 3'UTR ribosome occupancy in Rli1 depleted cells, indicating that Dom34 clears the bulk of unrecycled ribosomes. Thus, Rli1 is crucial for ribosome recycling in vivo and controls ribosome homeostasis. 3'UTR translation occurs in wild-type cells as well, and observations of elevated 3'UTR ribosomes during stress suggest that modulating recycling and reinitiation is involved in responding to environmental changes.

High-precision analysis of translational pausing by ribosome profiling in bacteria lacking EFP.

Ribosome profiling is a powerful method for globally assessing the activity of ribosomes in a cell. Despite its application in many organisms, ribosome profiling studies in bacteria have struggled to obtain the resolution necessary to precisely define translational pauses. Here, we report improvements that yield much higher resolution in E. coli profiling data, enabling us to more accurately assess ribosome pausing and refine earlier studies of the impact of polyproline motifs on elongation. We comprehensively characterize pausing at proline-rich motifs in the absence of elongation factor EFP. We find that only a small fraction of genes with strong pausing motifs have reduced ribosome density downstream, and we identify features that explain this phenomenon. These features allow us to predict which proteins likely have reduced output in the efp-knockout strain.