scholarly journals Ribosomal stalk proteins RPLP1 and RPLP2 promote biogenesis of flaviviral and cellular multi-pass transmembrane proteins

2019 ◽  
Author(s):  
Rafael K. Campos ◽  
Sagara Wijeratne ◽  
Premal Shah ◽  
Mariano A. Garcia-Blanco ◽  
Shelton S. Bradrick
Keyword(s):  
Protein Stability ◽  
Transmembrane Protein ◽  
Meta Analysis ◽  
Ribosome Profiling ◽  
Small Subset ◽  
Ribosome Density ◽  
Cellular Translation ◽  
Cellular Mrnas ◽  
Ribosomal Stalk ◽  
Ribosome Pausing

ABSTRACTDengue virus (DENV) and other mosquito-borne flaviviruses are highly dependent on the ribosomal stalk proteins, RPLP1 and RPLP2 (RPLP1/2), for efficient infection. Here, we show that RPLP1/2 function to relieve ribosome pausing within the DENV envelope coding sequence, leading to enhanced protein stability. We used ribosome profiling to evaluate viral and cellular translation in RPLP1/2-depleted cells. This revealed that ribosomes pause in the sequence coding for the N-terminus of the envelope protein, immediately downstream of sequences encoding two adjacent transmembrane domains (TMDs). RPLP1/2 function to enhance ribosome elongation at this position and increase viral protein stability, possibly by improving co-translational folding of DENV proteins. We also analyzed the effects of RPLP1/2 depletion on cellular translation. We find that RPLP1/2 affects ribosome density for a small subset of cellular mRNAs. However, meta-analysis of ribosome positions on all cellular mRNAs revealed slightly increased accumulation of ribosomes downstream of start codons in RPLP1/2-depleted cells, suggesting that RPLP1/2 enhance elongation efficiency. Importantly, we found that ribosome density on mRNAs encoding multiple TMDs was disproportionately affected by RPLP1/2 knockdown, implying a role for RPLP1/2 in transmembrane protein biogenesis. Together, our findings reveal insights into the function of RPLP1/2 in DENV and cellular translation.

scholarly journals Ribosomal stalk proteins RPLP1 and RPLP2 promote biogenesis of flaviviral and cellular multi-pass transmembrane proteins

2020 ◽  
Vol 48 (17) ◽  
pp. 9872-9885
Author(s):  
Rafael K Campos ◽  
H R Sagara Wijeratne ◽  
Premal Shah ◽  
Mariano A Garcia-Blanco ◽  
Shelton S Bradrick
Keyword(s):  
Protein Stability ◽  
Transmembrane Protein ◽  
Ribosome Profiling ◽  
Small Subset ◽  
Ribosome Density ◽  
Protein Biogenesis ◽  
Cellular Translation ◽  
Cellular Mrnas ◽  
Ribosomal Stalk ◽  
Ribosome Pausing

Abstract The ribosomal stalk proteins, RPLP1 and RPLP2 (RPLP1/2), which form the ancient ribosomal stalk, were discovered decades ago but their functions remain mysterious. We had previously shown that RPLP1/2 are exquisitely required for replication of dengue virus (DENV) and other mosquito-borne flaviviruses. Here, we show that RPLP1/2 function to relieve ribosome pausing within the DENV envelope coding sequence, leading to enhanced protein stability. We evaluated viral and cellular translation in RPLP1/2-depleted cells using ribosome profiling and found that ribosomes pause in the sequence coding for the N-terminus of the envelope protein, immediately downstream of sequences encoding two adjacent transmembrane domains (TMDs). We also find that RPLP1/2 depletion impacts a ribosome density for a small subset of cellular mRNAs. Importantly, the polarity of ribosomes on mRNAs encoding multiple TMDs was disproportionately affected by RPLP1/2 knockdown, implying a role for RPLP1/2 in multi-pass transmembrane protein biogenesis. These analyses of viral and host RNAs converge to implicate RPLP1/2 as functionally important for ribosomes to elongate through ORFs encoding multiple TMDs. We suggest that the effect of RPLP1/2 at TMD associated pauses is mediated by improving the efficiency of co-translational folding and subsequent protein stability.

scholarly journals Identification of novel translated small ORFs in Escherichia coli using complementary ribosome profiling approaches

2021 ◽  
Author(s):  
Anne Stringer ◽  
Carol Smith ◽  
Kyle Mangano ◽  
Joseph T. Wade
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
High Sensitivity ◽  
Purifying Selection ◽  
Ribosome Profiling ◽  
Small Subset ◽  
Stop Codons ◽  
Small Proteins ◽  
Domains Of Life ◽  
Short Orfs

Small proteins of <51 amino acids are abundant across all domains of life but are often overlooked because their small size makes them difficult to predict computationally, and they are refractory to standard proteomic approaches. Ribosome profiling has been used to infer the existence of small proteins by detecting the translation of the corresponding open reading frames (ORFs). Detection of translated short ORFs by ribosome profiling can be improved by treating cells with drugs that stall ribosomes at specific codons. Here, we combine the analysis of ribosome profiling data for Escherichia coli cells treated with antibiotics that stall ribosomes at either start or stop codons. Thus, we identify ribosome-occupied start and stop codons with high sensitivity for ∼400 novel putative ORFs. The newly discovered ORFs are mostly short, with 365 encoding proteins of <51 amino acids. We validate translation of several selected short ORFs, and show that many likely encode unstable proteins. Moreover, we present evidence that most of the newly identified short ORFs are not under purifying selection, suggesting they do not impact cell fitness, although a small subset have the hallmarks of functional ORFs. IMPORTANCE Small proteins of <51 amino acids are abundant across all domains of life but are often overlooked because their small size makes them difficult to predict computationally, and they are refractory to standard proteomic approaches. Recent studies have discovered small proteins by mapping the location of translating ribosomes on RNA using a technique known as ribosome profiling. Discovery of translated sORFs using ribosome profiling can be improved by treating cells with drugs that trap initiating ribosomes. Here, we show that combining these data with equivalent data for cells treated with a drug that stalls terminating ribosomes facilitates the discovery of small proteins. We use this approach to discover 365 putative genes that encode small proteins in Escherichia coli .

scholarly journals Dengue Virus Utilizes a Novel Strategy for Translation Initiation When Cap-Dependent Translation Is Inhibited

2006 ◽  
Vol 80 (6) ◽  
pp. 2976-2986 ◽  
Author(s):  
Dianna Edgil ◽  
Charlotta Polacek ◽  
Eva Harris
Keyword(s):  
Dengue Virus ◽  
Translation Initiation ◽  
Initiation Factor ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Eukaryotic Initiation Factor 4E ◽  
Cellular Translation ◽  
Cellular Mrnas ◽  
Novel Strategy

ABSTRACT Viruses have developed numerous mechanisms to usurp the host cell translation apparatus. Dengue virus (DEN) and other flaviviruses, such as West Nile and yellow fever viruses, contain a 5′ m7GpppN-capped positive-sense RNA genome with a nonpolyadenylated 3′ untranslated region (UTR) that has been presumed to undergo translation in a cap-dependent manner. However, the means by which the DEN genome is translated effectively in the presence of capped, polyadenylated cellular mRNAs is unknown. This report demonstrates that DEN replication and translation are not affected under conditions that inhibit cap-dependent translation by targeting the cap-binding protein eukaryotic initiation factor 4E, a key regulator of cellular translation. We further show that under cellular conditions in which translation factors are limiting, DEN can alternate between canonical cap-dependent translation initiation and a noncanonical mechanism that appears not to require a functional m7G cap. This DEN noncanonical translation is not mediated by an internal ribosome entry site but requires the interaction of the DEN 5′ and 3′ UTRs for activity, suggesting a novel strategy for translation of animal viruses.

IFITM3 rs12252 T>C polymorphism is associated with the risk of severe influenza: a meta-analysis

2015 ◽  
Vol 143 (14) ◽  
pp. 2975-2984 ◽  
Author(s):  
Y. XUAN ◽  
L. N. WANG ◽  
W. LI ◽  
H. R. ZI ◽  
Y. GUO ◽  
...  
Keyword(s):  
Influenza A ◽  
Web Of Science ◽  
Transmembrane Protein ◽  
Influenza Infection ◽  
Meta Analysis ◽  
Severe Illness ◽  
Severe Influenza ◽  
Increased Risk ◽  
Interferon Pathway ◽  
Comprehensive Literature Search

SUMMARYThe interferon-inducible transmembrane protein 3 (IFITM3), as one of the key genes involved in the interferon pathway, is critical for defending the host against influenza virus, and the rs12252 T>C variant in IFITM3 might be associated with susceptibility to severe influenza. Owing to contradictory and inconclusive results, we performed a meta-analysis to assess the association between rs12252 T>C polymorphism and severe influenza risk. A comprehensive literature search up to 1 August 2014 was conducted in EMBASE, Pubmed, Web of Science, VIP, Wanfang and CNKI databases. Four eligible studies with a total of 445 influenza patients and 3396 controls were included in this meta-analysis. Overall, our results demonstrated a significant association between the IFITM3 rs12252 T>C polymorphism and influenza risk [C vs. T: odds ratio (OR) 1·68, 95% confidence interval (CI) 1·32–2·13; CC vs. CT+TT: OR 2·38, 95% CI 1·52–3·73; CC+CT vs. TT: OR 1·62, 95% CI 1·18–2·22]. Stratification by ethnicity indicated that the variant C allele was associated with an 88% increased risk of influenza in Asians (C vs. T: OR 1·88, 95% CI 1·34–2·62). Moreover, subjects carrying the variant C allele had an increased risk of developing severe illness upon influenza infection (C vs. T: OR 2·70, 95% CI 1·86–3·94). However, no significant association was observed in patients with mild infection (C vs. T: OR 1·26, 95% CI 0·93–1·71). Our meta-analysis suggests that IFITM3 rs12252 T>C polymorphism is significantly associated with increased risk of severe influenza but not with the chance of initial virus infection.

scholarly journals Gi proteins mediate activation of the canonical hedgehog pathway in the myocardium

2014 ◽  
Vol 307 (1) ◽  
pp. H66-H72 ◽  
Author(s):  
Christian J. Carbe ◽  
Lan Cheng ◽  
Sankar Addya ◽  
Jessica I. Gold ◽  
Erhe Gao ◽  
...  
Keyword(s):  
Purinergic Signaling ◽  
Transmembrane Protein ◽  
Critical Role ◽  
Transcriptional Response ◽  
Neonatal Rat ◽  
Small Subset ◽  
Dependent Manner ◽  
Gli1 Expression ◽  
Ventricular Cardiomyocytes ◽  
First Time

During myocardial ischemia, upregulation of the hedgehog (Hh) pathway promotes neovascularization and increases cardiomyocyte survival. The canonical Hh pathway activates a transcriptional program through the Gli family of transcription factors by derepression of the seven-transmembrane protein smoothened (Smo). The mechanisms linking Smo to Gli are complex and, in some cell types, involve coupling of Smo to Gi proteins. In the present study, we investigated, for the first time, the transcriptional response of cardiomyocytes to sonic hedgehog (Shh) and the role of Gi protein utilization. Our results show that Shh strongly activates Gli1 expression by quantitative PCR in a Smo-dependent manner in neonatal rat ventricular cardiomyocytes. Microarray analysis of gene expression changes elicited by Shh and sensitive to a Smo inhibitor identified a small subset of 37 cardiomyocyte-specific genes regulated by Shh, including some in the PKA and purinergic signaling pathways. In addition, neonatal rat ventricular cardiomyocytes infected with an adenovirus encoding GiCT, a peptide that impairs receptor-Gi protein coupling, showed reduced activation of Hh targets. In vitro data were confirmed in transgenic mice with cardiomyocyte-inducible GiCT expression. Transgenic GiCT mice showed specific reduction of Gli1 expression in the heart under basal conditions and failed to upregulate the Hh pathway upon ischemia and reperfusion injury, unlike their littermate controls. This study characterizes, for the first time, the transcriptional response of cardiomyocytes to Shh and establishes a critical role for Smo coupling to Gi in Hh signaling in the normal and ischemic myocardium.

scholarly journals Inferring translational heterogeneity from ribosome profiling data

2019 ◽  
Author(s):  
Pedro do Couto Bordignon ◽  
Sebastian Pechmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Biosynthesis ◽  
Ribosome Profiling ◽  
Translation Regulation ◽  
Messenger Rnas ◽  
Translation Elongation ◽  
Cellular Homeostasis ◽  
Ribosome Density ◽  
Experimental Variability ◽  
Underlying Mechanisms

Translation of messenger RNAs into proteins by the ribosome is the most important step of protein biosynthesis. Accordingly, translation is tightly controlled and heavily regulated to maintain cellular homeostasis. Ribosome profiling (Ribo-seq) has revolutionized the study of translation by revealing many of its underlying mechanisms. However, equally many aspects of translation remain mysterious, in part also due to persisting challenges in the interpretation of data obtained from Ribo-seq experiments. Here, we show that some of the variability observed in Ribo-seq data has biological origins and reflects programmed heterogeneity of translation. To systematically identify sequences that are differentially translated (DT) across mRNAs beyond what can be attributed to experimental variability, we performed a comparative analysis of Ribo-seq data from Saccharomyces cerevisiae and derived a consensus ribosome density profile that reflects consistent signals in individual experiments. Remarkably, the thus identified DT sequences link to mechanisms known to regulate translation elongation and are enriched in genes important for protein and organelle biosynthesis. Our results thus highlight examples of translational heterogeneity that are encoded in the genomic sequences and tuned to optimizing cellular homeostasis. More generally, our work highlights the power of Ribo-seq to understand the complexities of translation regulation.

scholarly journals A meta-analysis of the activity, stability, and mutational characteristics of temperature-adapted enzymes

2021 ◽  
Author(s):  
Stewart Gault ◽  
Peter Higgins ◽  
Charles S Cockell ◽  
Kaitlyn Gillies
Keyword(s):  
Protein Stability ◽  
Enzyme Catalysis ◽  
Evolutionary Rate ◽  
Meta Analysis ◽  
Amino Acid Substitutions ◽  
Major Goal ◽  
Thermophilic Enzymes ◽  
Melting Temperatures ◽  
Psychrophilic Enzyme ◽  
Existing Data

Understanding the characteristics that define temperature-adapted enzymes has been a major goal of extremophile enzymology in recent decades. In this study, we explore these characteristics by comparing psychrophilic, mesophilic, and thermophilic enzymes. Through a meta-analysis of existing data, we show that psychrophilic enzymes exhibit a significantly larger gap (Tg) between their optimum and melting temperatures compared to mesophilic and thermophilic enzymes. These results suggest that Tg may be a useful indicator as to whether an enzyme is psychrophilic or not and that models of psychrophilic enzyme catalysis need to account for this gap. Additionally, by using predictive protein stability software, HoTMuSiC and PoPMuSiC, we show that the deleterious nature of amino acid substitutions to protein stability increases from psychrophiles to thermophiles. How this ultimately affects the mutational tolerance and evolutionary rate of temperature adapted organisms is currently unknown.

scholarly journals Understanding biases in ribosome profiling experiments reveals signatures of translation dynamics in yeast

2015 ◽  
Author(s):  
Jeffrey A Hussmann ◽  
Stephanie Patchett ◽  
Arlen Johnson ◽  
Sara Sawyer ◽  
William H Press
Keyword(s):  
Negative Correlation ◽  
Ribosome Profiling ◽  
Messenger Rnas ◽  
Translation Rate ◽  
Ribosome Density ◽  
Weak Negative Correlation ◽  
Specific Elongation ◽  
Positive Correlations ◽  
Cognate Trna

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.

Feasibility of developing a Twitter journal club for hematology/oncology education.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 11004-11004
Author(s):  
Elizabeth Henry ◽  
Devika Govind Das ◽  
Martina Cathryn Murphy
Keyword(s):  
Social Media ◽  
Journal Club ◽  
Meta Analysis ◽  
Small Subset ◽  
Tumor Type ◽  
Medical Trainees ◽  
Content Expert ◽  
Oncology Education ◽  
Social Media Platforms ◽  
Use Of Social Media

11004 Background: Medical trainees are increasingly utilizing social media platforms for professional development, networking and education. Twitter chats (TC) are a growing tool to engage health professionals in virtual multi-institutional, cross-discipline discussions. A meta-analysis of Twitter as a tool in residency education demonstrated high rates of satisfaction and concept retention. Despite rapid uptake, few studies address needs for social media use and implementation in graduate medical education. Methods: We created a Twitter account (@HOjournalclub) and registered a certified hashtag (#HOJournalClub) with healthcare symplur. For each monthly TC, a specific tumor type and relevant publication was selected. This information was disseminated and amplified to reach trainees on Twitter. A content expert was invited to each TC to provide additional commentary. During TCs, participants answer questions based on domains of critical journal appraisal. Qualitative and quantitative analysis was performed. Basic demographics and tracked hashtag use to measure impressions, participants, and tweets per TC were gathered. Responses were collated and general themes were assessed. Participants were surveyed on ease of participation, article accessibility, and prior use of social media for education. Results: Since inception, @HOJournalClub has grown to >650 followers. Most are US-based (83%) medical trainees or healthcare professionals. Additional followers are in South America, Africa, UK, Europe, Middle East, India, East Asia and Australia. Gender is evenly distributed (51% male, 49% female.) Five #HOJournalClub chats have been held to date. Each attracted a mean of 30 participants, generating a mean of 217 tweets. Chats garnered a mean of 270,000 impressions (221,000-319,000) in the 48h after TC. Most participants accessed the chat in real time, with a small subset responding at alternate times. This asynchronous use has enhanced international participation. In post-TC surveys, majority of respondents report being new (48%) or sporadic (48%) users of TCs. Survey participants reported TC participation increased interaction with others in the field, improved literature appraisal skills and led to changes in clinical practice. Conclusions: Implementation of a Twitter-based journal club is feasible and attracts participation from trainees, promoting engagement and networking. It represents a novel educational tool for engagement in multi-institutional, multi-national and cross-discipline discussion of relevant hematology/oncology literature.

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