核糖体图谱技术在植物学研究中的应用

随着高通量测序技术的持续发展和进步, 开发出很多新颖的测序技术, 为诸多悬而未决的生物学难题提供了解决方案。其中, 核糖体图谱技术能够在全基因组水平和单核苷酸分辨率上监控细胞内的翻译事件, 填补了转录组学和蛋白质组学研究之间的空隙。核糖体图谱技术不仅能够鉴定处于翻译状态的RNA分子, 还能够精确定位RNA分子上正在翻译的核苷酸, 进而准确描绘RNA分子上的开放阅读框。此外, 结合转录组测序数据, 核糖体图谱技术还可以确定每个转录本上的核糖体数量, 从而计算每个转录本的翻译效率。目前, 核糖体图谱技术已成功应用于动物、植物和微生物等研究领域, 加深了人们对翻译调控机制的认识。然而, 由于植物细胞和组织的特性, 核糖体图谱技术在植物学研究中的应用仍然存在局限。该文综述了核糖体图谱技术的实验原理, 以及在植物学研究中的相关进展。     

A Rapid Ribosome Profiling Method Elucidates Chloroplast Ribosome Behavior in Vivo

The profiling of ribosome footprints by deep sequencing has revolutionized the analysis of translation by mapping ribosomes with high resolution on a genome-wide scale. We present a variation on this approach that offers a rapid and cost-effective alternative for the genome-wide profiling of chloroplast ribosomes. Ribosome footprints from leaf tissue are hybridized to oligonucleotide tiling microarrays of the plastid ORFeome and report the abundance and translational status of every chloroplast mRNA. Each assay replaces several time-consuming traditional methods while also providing information that was previously inaccessible. To illustrate the utility of the approach, we show that it detects known defects in chloroplast gene expression in several nuclear mutants of maize (Zea mays) and that it reveals previously unsuspected defects. Furthermore, it provided firm answers to several lingering questions in chloroplast gene expression: (1) the overlapping atpB/atpE open reading frames, whose translation had been proposed to be coupled, are translated independently in vivo; (2) splicing is not a prerequisite for translation initiation on an intron-containing chloroplast RNA; and (3) a feedback control mechanism that links the synthesis of ATP synthase subunits in Chlamydomonas reinhardtii does not exist in maize. An analogous approach is likely to be useful for studies of mitochondrial gene expression.     

Modified ribosome profiling reveals high abundance of ribosome protected mRNA fragments derived from 3′ untranslated regions

Ribosome profiling identifies ribosome positions on translated mRNAs. A prominent feature of published datasets is the near complete absence of ribosomes in 3′ untranslated regions (3′UTR) although substantial ribosome density can be observed on non-coding RNAs. Here we perform ribosome profiling in cultured Drosophila and human cells and show that different features of translation are revealed depending on the nuclease and the digestion conditions used. Most importantly, we observe high abundance of ribosome protected fragments in 3′UTRs of thousands of genes without manipulation of translation termination. Affinity purification of ribosomes indicates that the 3′UTR reads originate from ribosome protected fragments. Association of ribosomes with the 3′UTR may be due to ribosome migration through the stop codon or 3′UTR mRNA binding to ribosomes on the coding sequence. This association depends primarily on the relative length of the 3′UTR and may be related to translational regulation or ribosome recycling, for which the efficiency is known to inversely correlate with 3′UTR length. Together our results indicate that ribosome profiling is highly dependent on digestion conditions and that ribosomes commonly associate with the 3′UTR, which may have a role in translational regulation.     

Translation inhibitors cause abnormalities in ribosome profiling experiments

Ribosome profiling and high-throughput sequencing provide unprecedented opportunities for the analysis of mRNA translation. Using this novel method, several studies have demonstrated the widespread role of short upstream reading frames in translational control as well as slower elongation at the beginning of open reading frames in response to stress. Based on the initial studies, the importance of adding or omitting translation inhibitors, such as cycloheximide, was noted as it markedly affected ribosome coverage profiles. For that reason, many recent studies omitted translation inhibitors in the culture medium. Here, we investigate the influence of ranging cycloheximide concentrations on ribosome profiles in Saccharomyces cerevisiae and demonstrate that increasing the drug concentration can overcome some of the artifacts. We subjected cells to various manipulations and show that neither oxidative stress nor heat shock nor amino acid starvation affect translation elongation. Instead, the observations in the initial studies are the result of cycloheximide-inflicted artifacts. Likewise, we find little support for short upstream reading frames to be involved in widespread protein synthesis regulation under stress conditions. Our study highlights the need for better standardization of ribosome profiling methods.     

Ribosomes bind leaderless mRNA in Escherichia coli through recognition of their 5'-terminal AUG

Leaderless mRNAs are translated in the absence of upstream signals that normally contribute to ribosome binding and translation efficiency. In order to identify ribosomal components that interact with leaderless mRNA, a fragment of leaderless cI mRNA from bacteriophage λ, with a 4-thiouridine (4^S-U) substituted at the +2 position of the AUG start codon, was used to form cross-links to Escherichia coli ribosomes during binary (mRNA+ribosome) and ternary (mRNA+ribosome+initiator tRNA) complex formation. Ribosome binding assays (i.e., toeprints) demonstrated tRNA-dependent binding of leaderless mRNA to ribosomes; however, cross-links between the start codon and 30S subunit rRNA and r-proteins formed independent of initiator tRNA. Toeprints revealed that a leaderless mRNA's 5′-AUG is required for stable binding. Furthermore, the addition of a 5′-terminal AUG triplet to a random RNA fragment can make it both competent and competitive for ribosome binding, suggesting that a leaderless mRNA's start codon is a major feature for ribosome interaction. Cross-linking assays indicate that a subset of 30S subunit r-proteins, located at either end of the mRNA tunnel, contribute to tRNA-independent contacts and/or interactions with a leaderless mRNA's start codon. The interaction of leaderless mRNA with ribosomes may reveal features of mRNA binding and AUG recognition that are distinct from known signals but are important for translation initiation of all mRNAs.     

Identification of internal ribosome entry segment (IRES)-trans-acting factors for the Myc family of IRESs

The proto-oncogenes c-, L-, and N-myc can all be translated by the alternative method of internal ribosome entry whereby the ribosome is recruited to a complex structural element (an internal ribosome entry segment [IRES]). Ribosome recruitment is dependent upon the presence of IRES-trans-acting factors (ITAFs) that act as RNA chaperones and allow the mRNA to attain the correct conformation for the interaction of the 40S subunit. One of the major challenges for researchers in this area is to determine whether there are groups of ITAFs that regulate the IRES-mediated translation of subsets of mRNAs. We have identified four proteins, termed GRSF-1 (G-rich RNA sequence binding factor 1), YB-1 (Y-box binding protein 1), PSF (polypyrimidine tract binding protein-associated splicing factor), and its binding partner, p54nrb, that bind to the myc family of IRESs. We show that these proteins positively regulate the translation of the Myc family of oncoproteins (c-, L-, and N-Myc) in vivo and in vitro. Interestingly, synthesis from the unrelated IRESs, BAG-1 and Apaf-1, was not affected by YB-1, GRSF-1, or PSF levels in vivo, suggesting that these three ITAFs are specific to the myc IRESs. Myc proteins play a role in cell proliferation; therefore, these results have important implications regarding the control of tumorigenesis.     

Translational control of coronaviruses

Coronaviruses represent a large family of enveloped RNA viruses that infect a large spectrum of animals. In humans, the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic and is genetically related to SARS-CoV and Middle East respiratory syndrome-related coronavirus (MERS-CoV), which caused outbreaks in 2002 and 2012, respectively. All viruses described to date entirely rely on the protein synthesis machinery of the host cells to produce proteins required for their replication and spread. As such, virus often need to control the cellular translational apparatus to avoid the first line of the cellular defense intended to limit the viral propagation. Thus, coronaviruses have developed remarkable strategies to hijack the host translational machinery in order to favor viral protein production. In this review, we will describe some of these strategies and will highlight the role of viral proteins and RNAs in this process.     

Translation Initiation at the CUU Codon Is Mediated by the Internal Ribosome Entry Site of an Insect Picorna-Like Virus In Vitro

AUG-unrelated translation initiation was found in an insect picorna-like virus, Plautia stali intestine virus (PSIV). The positive-strand RNA genome of the virus contains two nonoverlapping open reading frames (ORFs). The capsid protein gene is located in the 3′-proximal ORF and lacks an AUG initiation codon. We examined the translation mechanism and the initiation codon of the capsid protein gene by using various dicistronic and monocistronic RNAs in vitro. The capsid protein gene was translated cap independently in the presence of the upstream cistron, indicating that the gene is translated by internal ribosome entry. Deletion analysis showed that the internal ribosome entry site (IRES) consisted of approximately 250 bases and that its 3′ boundary extended slightly into the capsid-coding region. The initiation codon for the IRES-mediated translation was identified as the CUU codon, which is located just upstream of the 5′ terminus of the capsid-coding region by site-directed mutagenesis. In vitro translation assays of monocistronic RNAs lacking the 5′ part of the IRES showed that this CUU codon was not recognized by scanning ribosomes. This suggests that the PSIV IRES can effectively direct translation initiation without stable codon-anticodon pairing between the initiation codon and the initiator methionyl-tRNA.     

Ribosome collisions and translation efficiency: optimization by codon usage and mRNA destabilization

Individual mRNAs are translated by multiple ribosomes that initiate translation with an interval of a few seconds. The ribosome speed is codon dependent, and ribosome queuing has been suggested to explain specific data for translation of some mRNAs in vivo. By modeling the stochastic translation process as a traffic problem, we here analyze conditions and consequences of collisions and queuing. The model allowed us to determine the on-rate (0.8 to 1.1 initiations/s) and the time (1 s) the preceding ribosome occludes initiation for Escherichia coli lacZ mRNA in vivo. We find that ribosome collisions and queues are inevitable consequences of a stochastic translation mechanism that reduce the translation efficiency substantially on natural mRNAs. The cells minimize collisions by having its mRNAs being unstable and by a highly selected codon usage in the start of the mRNA. The cost of mRNA breakdown is offset by the concomitant increase in translation efficiency.     

Switch from Cap- to Factorless IRES-Dependent 0 and +1 Frame Translation during Cellular Stress and Dicistrovirus Infection

Internal ribosome entry sites (IRES) are utilized by a subset of cellular and viral mRNAs to initiate translation during cellular stress and virus infection when canonical cap-dependent translation is compromised. The intergenic region (IGR) IRES of the Dicistroviridae uses a streamlined mechanism in which it can directly recruit the ribosome in the absence of initiation factors and initiates translation using a non-AUG codon. A subset of IGR IRESs including that from the honey bee viruses can also direct translation of an overlapping +1 frame gene. In this study, we systematically examined cellular conditions that lead to IGR IRES-mediated 0 and +1 frame translation in Drosophila S2 cells. Towards this, a novel bicistronic reporter that exploits the 2A “stop-go” peptide was developed to allow the detection of IRES-mediated translation in vivo. Both 0 and +1 frame translation by the IGR IRES are stimulated under a number of cellular stresses and in S2 cells infected by cricket paralysis virus, demonstrating a switch from cap-dependent to IRES-dependent translation. The regulation of the IGR IRES mechanism ensures that both 0 frame viral structural proteins and +1 frame ORFx protein are optimally expressed during virus infection.     

Understanding Biases in Ribosome Profiling Experiments Reveals Signatures of Translation Dynamics in Yeast

Ribosome profiling produces snapshots of the locations of actively translating ribosomes on messenger RNAs. These snapshots can be used to make inferences about translation dynamics. Recent ribosome profiling studies in yeast, however, have reached contradictory conclusions regarding the average translation rate of each codon. Some experiments have used cycloheximide (CHX) to stabilize ribosomes before measuring their positions, and these studies all counterintuitively report a weak negative correlation between the translation rate of a codon and the abundance of its cognate tRNA. In contrast, some experiments performed without CHX report strong positive correlations. To explain this contradiction, we identify unexpected patterns in ribosome density downstream of each type of codon in experiments that use CHX. These patterns are evidence that elongation continues to occur in the presence of CHX but with dramatically altered codon-specific elongation rates. The measured positions of ribosomes in these experiments therefore do not reflect the amounts of time ribosomes spend at each position in vivo. These results suggest that conclusions from experiments in yeast using CHX may need reexamination. In particular, we show that in all such experiments, codons decoded by less abundant tRNAs were in fact being translated more slowly before the addition of CHX disrupted these dynamics.     

Unique and conserved features of genome and proteome of SARS-coronavirus,an early split-off from the coronavirus group 2 lineage

The genome organization and expression strategy of the newly identified severe acute respiratory syndrome coronavirus (SARS-CoV) were predicted using recently published genome sequences. Fourteen putative open reading frames were identified, 12 of which were predicted to be expressed from a nested set of eight subgenomic mRNAs. The synthesis of these mRNAs in SARS-CoV-infected cells was confirmed experimentally. The 4382- and 7073 amino acid residue SARS-CoV replicase polyproteins are predicted to be cleaved into 16 subunits by two viral proteinases (bringing the total number of SARS-CoV proteins to 28). A phylogenetic analysis of the replicase gene, using a distantly related torovirus as an outgroup, demonstrated that, despite a number of unique features, SARS-CoV is most closely related to group 2 coronaviruses. Distant homologs of cellular RNA processing enzymes were identified in group 2 coronaviruses, with four of them being conserved in SARS-CoV. These newly recognized viral enzymes place the mechanism of coronavirus RNA synthesis in a completely new perspective. Furthermore, together with previously described viral enzymes, they will be important targets for the design of antiviral strategies aimed at controlling the further spread of SARS-CoV.     

The 3′-Untranslated Region of Hepatitis C Virus RNA Enhances Translation from an Internal Ribosomal Entry Site

Translation of most eukaryotic mRNAs and many viral RNAs is enhanced by their poly(A) tails. Hepatitis C virus (HCV) contains a positive-stranded RNA genome which does not have a poly(A) tail but has a stretch of 98 nucleotides (X region) at the 3′-untranslated region (UTR), which assumes a highly conserved stem-loop structure. This X region binds a polypyrimidine tract-binding protein (PTB), which also binds to the internal ribosome entry site (IRES) in HCV 5′-UTR. These RNA-protein interactions may regulate its translation. We generated a set of HCV RNAs differing only in their 3′-UTRs and compared their translation efficiencies. HCV RNA containing the X region was translated three- to fivefold more than the corresponding RNAs without this region. Mutations that abolished PTB binding in the X region reduced, but did not completely abolish, enhancement in translation. The X region also enhanced translation from another unrelated IRES (from encephalomyocarditis virus RNA), but did not affect the 5′-end-dependent translation of globin mRNA in either monocistronic or bicistronic RNAs. It did not appear to affect RNA stability. The free X region added in trans, however, did not enhance translation, indicating that the translational enhancement by the X region occurs only in cis. These results demonstrate that the highly conserved 3′ end of HCV RNA provides a novel mechanism for enhancement of HCV translation and may offer a target for antiviral agents.     

Coronaviruses: Important Emerging Human Pathogens

The identification of Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 reaffirmed the importance of understanding how coronaviruses emerge, infect, and cause disease. By comparing what is known about severe acute respiratory syndrome coronavirus (SARS-CoV) to what has recently been found for MERS-CoV, researchers are discovering similarities and differences that may be important for pathogenesis. Here we discuss what is known about each virus and what gaps remain in our understanding, especially concerning MERS-CoV.