Control of translation during the unfolded protein response in maize seedlings: Life without PERKs

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) defines a condition called ER stress that induces the unfolded protein response (UPR). The UPR in mammalian cells attenuates protein synthesis initiation, which prevents the piling up of misfolded proteins in the ER. Mammalian cells rely on Protein Kinase RNA‐Like Endoplasmic Reticulum Kinase (PERK) phosphorylation of eIF2α to arrest protein synthesis, however, plants do not have a PERK homolog, so the question is whether plants control translation in response to ER stress. We compared changes in RNA levels in the transcriptome to the RNA levels protected by ribosomes and found a decline in translation efficiency, including many UPR genes, in response to ER stress. The decline in translation efficiency is due to the fact that many mRNAs are not loaded onto polyribosomes (polysomes) in proportion to their increase in total RNA, instead some of the transcripts accumulate in stress granules (SGs). The RNAs that populate SGs are not derived from the disassembly of polysomes because protein synthesis remains steady during stress. Thus, the surge in transcription of UPR genes in response to ER stress is accompanied by the formation of SGs, and the sequestration of mRNAs in SGs may serve to temporarily relieve the translation load during ER stress

A rapid and simple quantitative method for specific Detection of Smaller Co-terminal RNA by PCR (DeSCo-PCR): Application to the detection of viral subgenomic RNAs

RNAs that are 5’-truncated versions of a longer RNA, but share the same 3’ terminus can be generated by alternative promoters in transcription of cellular mRNAs or by replicating RNA viruses. These truncated RNAs cannot be distinguished from the longer RNA by a simple two-primer RT-PCR because primers that anneal to the cDNA from the smaller RNA also anneal to - and amplify - the longer RNA-derived cDNA. Thus, laborious methods, such as northern blot hybridization, are used to distinguish shorter from longer RNAs. For rapid, low-cost and specific detection of these truncated RNAs, we report Detection of Smaller Co-terminal RNA by PCR (DeSCo-PCR). DeSCo-PCR employs a non-extendable blocking primer (BP), which outcompetes a forward primer (FP) for annealing to longer RNA-derived cDNA, while FP outcompetes BP for annealing to shorter RNA-derived cDNA. In the presence of BP, FP and the reverse primer, only cDNA from the shorter RNA is amplified in a single-tube reaction containing both RNAs. Many positive strand RNA viruses generate 5’-truncated forms of the genomic RNA (gRNA) called subgenomic RNAs (sgRNA), which play key roles in viral gene expression and pathogenicity. We demonstrate that DeSCo-PCR is easily optimized to selectively detect relative quantities of sgRNAs of red clover necrotic mosaic virus from plants and Zika virus from human cells, each infected with viral strains that generate different amounts of sgRNA. This technique should be readily adaptable to other sgRNA-producing viruses, and for quantitative detection of any truncated or alternatively spliced RNA

Global effects of plant virus infection, viral noncoding RNAs, and unfolded protein response on plant gene expression

Plants are subject to a variety of abiotic and biotic stresses, including virus infection. This leads to enormous losses in crop yield and quality worldwide. Understanding how plants respond to these stresses can enable researchers to develop more healthy and robust plant varieties. The main objective of my research is to explore (i) the transcriptional and translational control of cellular gene expression in response to virus infection and (ii) the role(s) of viral noncoding subgenomic (ncsg)RNAs during infection. For this, I used red clover necrotic mosaic virus (RCNMV) as a model for economically important Tombusvirids. RCNMV generates a 3’ coterminal viral ncsgRNA, called SR1f, that belongs to the class of viral subgenomic (sg)RNAs that are functional in human flavivirus pathogenesis but which are still understudied in plant virus infection. Additionally, I also explored how translation is regulated in plants during unfolded protein response (UPR), which is elicited by many viruses and abiotic stresses. A prerequisite for investigating viral sgRNAs is an RNA detection method that can distinguish between the coterminal genomic and sgRNAs. Using RCNMV SR1f and the analogous ncsgRNA from Zika virus (sfRNA), I developed a novel RT-PCR-based method, called DeSCo-PCR (Detection of smaller coterminal RNAs by PCR), for simple, quick, quantitative, and specific detection of viral sgRNAs. I demonstrate its advantages over the traditionally-used northern blot hybridization for detecting viral sgRNAs. This is the first RT-PCR method that distinguishes genomic from sgRNAs in most positive-sense RNA viruses. Next, I wanted to assess the role(s) of RCNMV SR1f during infection. RCNMV SR1f belongs to the class of exoribonuclease-resistant (xr)RNA-derived viral ncsgRNAs. In plants, viral ncsgRNAs play a role in determining the severity of symptoms and the success of infection. Therefore, to explore the functions and effects of SR1f, I (i) used RNA sequencing (RNA-seq) to compare how infection with RCNMV constructs, which can or cannot produce SR1f, affect the transcriptomes of Nicotiana benthamiana and RCNMV, (ii) assessed the role of SR1f in counteracting the antiviral RNA silencing response in Arabidopsis thaliana, and (iii) determined the requirement of XRN4 for generating RCNMV SR1f in A. thaliana. Next, I used ribosome profiling (Ribo-seq) to assess how host and viral genes are translationally regulated in RCNMV-infected plants. Most genome-wide host-virus interaction studies have used RNA-seq, which does not provide any information on translational control. Translational control is a tightly-regulated process that provides a more rapid change in gene expression than a transcriptional response. Furthermore, viruses rely completely on cellular translation machinery for viral protein synthesis. However, translational control during plant virus-host interaction has rarely been studied at the genome-wide level. Therefore, I used Ribo-seq to (i) assess the effects of RCNMV infection on the transcriptome and the translatome of A. thaliana at early and late stages of infection, (ii) identify cellular genes that are transcriptionally and translationally-regulated in response to virus infection, and (iii) assess the translational landscape of RCNMV mRNAs in infected cells. Finally, I also used Ribo-seq to assess the translational control in roots of Zea mays seedlings during UPR. The PKR-like ER kinase (PERK)-mediated UPR pathway, which results in phosphorylation of eIF2α and subsequent inhibition of global translation in mammalian cells, is absent in the plant system. Therefore, I wanted to determine if translational control is as important in plants as it is in mammalian cells during UPR. I used Ribo-seq and other molecular assays to (i) determine if there is global inhibition of translation in plants during UPR, (ii) calculate the translational efficiencies of several UPR-responsive mRNAs, and (iii) determine the fate of the UPR-responsive mRNAs that were transcriptionally upregulated during UPR

Global effects of plant virus infection, viral noncoding RNAs, and unfolded protein response on plant gene expression

Plants are subject to a variety of abiotic and biotic stresses, including virus infection. This leads to enormous losses in crop yield and quality worldwide. Understanding how plants respond to these stresses can enable researchers to develop more healthy and robust plant varieties. The main objective of my research is to explore (i) the transcriptional and translational control of cellular gene expression in response to virus infection and (ii) the role(s) of viral noncoding subgenomic (ncsg)RNAs during infection. For this, I used red clover necrotic mosaic virus (RCNMV) as a model for economically important Tombusvirids. RCNMV generates a 3’ coterminal viral ncsgRNA, called SR1f, that belongs to the class of viral subgenomic (sg)RNAs that are functional in human flavivirus pathogenesis but which are still understudied in plant virus infection. Additionally, I also explored how translation is regulated in plants during unfolded protein response (UPR), which is elicited by many viruses and abiotic stresses. A prerequisite for investigating viral sgRNAs is an RNA detection method that can distinguish between the coterminal genomic and sgRNAs. Using RCNMV SR1f and the analogous ncsgRNA from Zika virus (sfRNA), I developed a novel RT-PCR-based method, called DeSCo-PCR (Detection of smaller coterminal RNAs by PCR), for simple, quick, quantitative, and specific detection of viral sgRNAs. I demonstrate its advantages over the traditionally-used northern blot hybridization for detecting viral sgRNAs. This is the first RT-PCR method that distinguishes genomic from sgRNAs in most positive-sense RNA viruses. Next, I wanted to assess the role(s) of RCNMV SR1f during infection. RCNMV SR1f belongs to the class of exoribonuclease-resistant (xr)RNA-derived viral ncsgRNAs. In plants, viral ncsgRNAs play a role in determining the severity of symptoms and the success of infection. Therefore, to explore the functions and effects of SR1f, I (i) used RNA sequencing (RNA-seq) to compare how infection with RCNMV constructs, which can or cannot produce SR1f, affect the transcriptomes of Nicotiana benthamiana and RCNMV, (ii) assessed the role of SR1f in counteracting the antiviral RNA silencing response in Arabidopsis thaliana, and (iii) determined the requirement of XRN4 for generating RCNMV SR1f in A. thaliana. Next, I used ribosome profiling (Ribo-seq) to assess how host and viral genes are translationally regulated in RCNMV-infected plants. Most genome-wide host-virus interaction studies have used RNA-seq, which does not provide any information on translational control. Translational control is a tightly-regulated process that provides a more rapid change in gene expression than a transcriptional response. Furthermore, viruses rely completely on cellular translation machinery for viral protein synthesis. However, translational control during plant virus-host interaction has rarely been studied at the genome-wide level. Therefore, I used Ribo-seq to (i) assess the effects of RCNMV infection on the transcriptome and the translatome of A. thaliana at early and late stages of infection, (ii) identify cellular genes that are transcriptionally and translationally-regulated in response to virus infection, and (iii) assess the translational landscape of RCNMV mRNAs in infected cells. Finally, I also used Ribo-seq to assess the translational control in roots of Zea mays seedlings during UPR. The PKR-like ER kinase (PERK)-mediated UPR pathway, which results in phosphorylation of eIF2α and subsequent inhibition of global translation in mammalian cells, is absent in the plant system. Therefore, I wanted to determine if translational control is as important in plants as it is in mammalian cells during UPR. I used Ribo-seq and other molecular assays to (i) determine if there is global inhibition of translation in plants during UPR, (ii) calculate the translational efficiencies of several UPR-responsive mRNAs, and (iii) determine the fate of the UPR-responsive mRNAs that were transcriptionally upregulated during UPR

Global effects of plant virus infection, viral noncoding RNAs, and unfolded protein response on plant gene expression

Plants are subject to a variety of abiotic and biotic stresses, including virus infection. This leads to enormous losses in crop yield and quality worldwide. Understanding how plants respond to these stresses can enable researchers to develop more healthy and robust plant varieties. The main objective of my research is to explore (i) the transcriptional and translational control of cellular gene expression in response to virus infection and (ii) the role(s) of viral noncoding subgenomic (ncsg)RNAs during infection. For this, I used red clover necrotic mosaic virus (RCNMV) as a model for economically important Tombusvirids. RCNMV generates a 3’ coterminal viral ncsgRNA, called SR1f, that belongs to the class of viral subgenomic (sg)RNAs that are functional in human flavivirus pathogenesis but which are still understudied in plant virus infection. Additionally, I also explored how translation is regulated in plants during unfolded protein response (UPR), which is elicited by many viruses and abiotic stresses. A prerequisite for investigating viral sgRNAs is an RNA detection method that can distinguish between the coterminal genomic and sgRNAs. Using RCNMV SR1f and the analogous ncsgRNA from Zika virus (sfRNA), I developed a novel RT-PCR-based method, called DeSCo-PCR (Detection of smaller coterminal RNAs by PCR), for simple, quick, quantitative, and specific detection of viral sgRNAs. I demonstrate its advantages over the traditionally-used northern blot hybridization for detecting viral sgRNAs. This is the first RT-PCR method that distinguishes genomic from sgRNAs in most positive-sense RNA viruses. Next, I wanted to assess the role(s) of RCNMV SR1f during infection. RCNMV SR1f belongs to the class of exoribonuclease-resistant (xr)RNA-derived viral ncsgRNAs. In plants, viral ncsgRNAs play a role in determining the severity of symptoms and the success of infection. Therefore, to explore the functions and effects of SR1f, I (i) used RNA sequencing (RNA-seq) to compare how infection with RCNMV constructs, which can or cannot produce SR1f, affect the transcriptomes of Nicotiana benthamiana and RCNMV, (ii) assessed the role of SR1f in counteracting the antiviral RNA silencing response in Arabidopsis thaliana, and (iii) determined the requirement of XRN4 for generating RCNMV SR1f in A. thaliana. Next, I used ribosome profiling (Ribo-seq) to assess how host and viral genes are translationally regulated in RCNMV-infected plants. Most genome-wide host-virus interaction studies have used RNA-seq, which does not provide any information on translational control. Translational control is a tightly-regulated process that provides a more rapid change in gene expression than a transcriptional response. Furthermore, viruses rely completely on cellular translation machinery for viral protein synthesis. However, translational control during plant virus-host interaction has rarely been studied at the genome-wide level. Therefore, I used Ribo-seq to (i) assess the effects of RCNMV infection on the transcriptome and the translatome of A. thaliana at early and late stages of infection, (ii) identify cellular genes that are transcriptionally and translationally-regulated in response to virus infection, and (iii) assess the translational landscape of RCNMV mRNAs in infected cells. Finally, I also used Ribo-seq to assess the translational control in roots of Zea mays seedlings during UPR. The PKR-like ER kinase (PERK)-mediated UPR pathway, which results in phosphorylation of eIF2α and subsequent inhibition of global translation in mammalian cells, is absent in the plant system. Therefore, I wanted to determine if translational control is as important in plants as it is in mammalian cells during UPR. I used Ribo-seq and other molecular assays to (i) determine if there is global inhibition of translation in plants during UPR, (ii) calculate the translational efficiencies of several UPR-responsive mRNAs, and (iii) determine the fate of the UPR-responsive mRNAs that were transcriptionally upregulated during UPR

Effects of the Noncoding Subgenomic RNA of Red Clover Necrotic Mosaic Virus in Virus Infection

In recent years, a new class of viral noncoding subgenomic RNA (ncsgRNA) has been identified. This RNA is generated as a stable degradation product via an exoribonuclease-resistant RNA (xrRNA) structure, which blocks the progression of 5′→3′ exoribonuclease on viral RNAs in infected cells. Here, we assess the effects of the ncsgRNA of red clover necrotic mosaic virus (RCNMV), called SR1f, in infected plants. We demonstrate the following: (i) the absence of SR1f reduces symptoms and decreases viral RNA accumulation in Nicotiana benthamiana and Arabidopsis thaliana plants; (ii) SR1f has an essential function other than suppression of RNA silencing; and (iii) the cytoplasmic exoribonuclease involved in mRNA turnover, XRN4, is not required for SR1f production or virus infection. A comparative transcriptomic analysis in N. benthamiana infected with wild-type RCNMV or an SR1f-deficient mutant RCNMV revealed that wild-type RCNMV infection, which produces SR1f and much higher levels of virus, has a greater and more significant impact on cellular gene expression than the SR1f-deficient mutant. Upregulated pathways include plant hormone signaling, plant-pathogen interaction, MAPK signaling, and several metabolic pathways, while photosynthesis-related genes were downregulated. We compare this to host genes known to participate in infection by other tombusvirids. Viral reads revealed a 10- to 100-fold ratio of positive to negative strand, and the abundance of reads of both strands mapping to the 3′ region of RCNMV RNA1 support the premature transcription termination mechanism of synthesis for the coding sgRNA. These results provide a framework for future studies of the interactions and functions of noncoding RNAs of plant viruses.This article is published as Kanodia, Pulkit, and W. Allen Miller. "Effects of the noncoding subgenomic RNA of red clover necrotic mosaic virus in virus infection." Journal of virology 96 (2022): e01815-21. doi:10.1128/jvi.01815-21. Posted with permission. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license

Variational inference for detecting differential translation in ribosome profiling studies

Translational efficiency change is an important mechanism for regulating protein synthesis. Experiments with paired ribosome profiling (Ribo-seq) and mRNA-sequencing (RNA-seq) allow the study of translational efficiency by simultaneously quantifying the abundances of total transcripts and those that are being actively translated. Existing methods for Ribo-seq data analysis either ignore the pairing structure in the experimental design or treat the paired samples as fixed effects instead of random effects. To address these issues, we propose a hierarchical Bayesian generalized linear mixed effects model which incorporates a random effect for the paired samples according to the experimental design. We provide an analytical software tool, “riboVI,” that uses a novel variational Bayesian algorithm to fit our model in an efficient way. Simulation studies demonstrate that “riboVI” outperforms existing methods in terms of both ranking differentially translated genes and controlling false discovery rate. We also analyzed data from a real ribosome profiling experiment, which provided new biological insight into virus-host interactions by revealing changes in hormone signaling and regulation of signal transduction not detected by other Ribo-seq data analysis tools.This article is published as Walker DC, Lozier ZR, Bi R, Kanodia P, Miller WA and Liu P (2023) Variational inference for detecting differential translation in ribosome profiling studies. Front. Genet. 14:1178508. doi: 10.3389/fgene.2023.1178508. Posted with permission. © 2023 Walker, Lozier, Bi, Kanodia, Miller and Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms

Control of translation during the unfolded protein response in maize seedlings: Life without PERKs

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) defines a condition called ER stress that induces the unfolded protein response (UPR). The UPR in mammalian cells attenuates protein synthesis initiation, which prevents the piling up of misfolded proteins in the ER. Mammalian cells rely on Protein Kinase RNA‐Like Endoplasmic Reticulum Kinase (PERK) phosphorylation of eIF2α to arrest protein synthesis, however, plants do not have a PERK homolog, so the question is whether plants control translation in response to ER stress. We compared changes in RNA levels in the transcriptome to the RNA levels protected by ribosomes and found a decline in translation efficiency, including many UPR genes, in response to ER stress. The decline in translation efficiency is due to the fact that many mRNAs are not loaded onto polyribosomes (polysomes) in proportion to their increase in total RNA, instead some of the transcripts accumulate in stress granules (SGs). The RNAs that populate SGs are not derived from the disassembly of polysomes because protein synthesis remains steady during stress. Thus, the surge in transcription of UPR genes in response to ER stress is accompanied by the formation of SGs, and the sequestration of mRNAs in SGs may serve to temporarily relieve the translation load during ER stress.This article is published as Kanodia, Pulkit, Paramasivan Vijayapalani, Renu Srivastava, Ran Bi, Peng Liu, W. Allen Miller, and Stephen H. Howell. "Control of translation during the unfolded protein response in maize seedlings: Life without PERKs." Plant Direct 4, no. 7 (2020): e00241. doi: 10.1002/pld3.241.</p

A rapid and simple quantitative method for specific Detection of Smaller Co-terminal RNA by PCR (DeSCo-PCR): Application to the detection of viral subgenomic RNAs

RNAs that are 5’-truncated versions of a longer RNA, but share the same 3’ terminus can be generated by alternative promoters in transcription of cellular mRNAs or by replicating RNA viruses. These truncated RNAs cannot be distinguished from the longer RNA by a simple two-primer RT-PCR because primers that anneal to the cDNA from the smaller RNA also anneal to - and amplify - the longer RNA-derived cDNA. Thus, laborious methods, such as northern blot hybridization, are used to distinguish shorter from longer RNAs. For rapid, low-cost and specific detection of these truncated RNAs, we report Detection of Smaller Co-terminal RNA by PCR (DeSCo-PCR). DeSCo-PCR employs a non-extendable blocking primer (BP), which outcompetes a forward primer (FP) for annealing to longer RNA-derived cDNA, while FP outcompetes BP for annealing to shorter RNA-derived cDNA. In the presence of BP, FP and the reverse primer, only cDNA from the shorter RNA is amplified in a single-tube reaction containing both RNAs. Many positive strand RNA viruses generate 5’-truncated forms of the genomic RNA (gRNA) called subgenomic RNAs (sgRNA), which play key roles in viral gene expression and pathogenicity. We demonstrate that DeSCo-PCR is easily optimized to selectively detect relative quantities of sgRNAs of red clover necrotic mosaic virus from plants and Zika virus from human cells, each infected with viral strains that generate different amounts of sgRNA. This technique should be readily adaptable to other sgRNA-producing viruses, and for quantitative detection of any truncated or alternatively spliced RNA.This is a manuscript of an article published as Kanodia, Pulkit, K. Reddisiva Prasanth, Vicky C. Roa-Linares, Shelton S. Bradrick, Mariano A. Garcia-Blanco, and W. Allen Miller. "A rapid and simple quantitative method for specific Detection of Smaller Co-terminal RNA by PCR (DeSCo-PCR): Application to the detection of viral subgenomic RNAs." RNA (2020). doi: 10.1261/rna.074963.120.</p