Stalling Events Research Articles

199 Awardee Talk: Insights into translational regulation through tRNA sequencing, RNA sequencing, and ribosome profiling

Abstract Translation acts as an additional layer of regulation that has an important role in gene expression and function. Due to a phenomenon called codon usage bias, highly expressed genes are thought to be codon-biased to support efficient translation. As more than one codon can code for the same amino acid (synonymous codons), organisms may exhibit preferences for specific codons that facilitate increased expression of important genes due to variations in the availability of corresponding tRNAs. Advances in sequencing technologies have recently permitted the study of the translatome, which refers to the entire population of mRNA associated with ribosomes for protein synthesis and can be investigated through ribosome profiling. This cutting-edge technique allows the identification of actively translated regions, but can also reveal translational pausing events that stem from the presence of SNPs. Our previous research has demonstrated variation in tRNA expression across tissues and different states of health, leading us to consider the connection between tRNA abundance and translational stalling due to SNPs. By coupling ribosome profiling with tRNA sequencing and RNA sequencing, we have investigated all elements of translational machinery to predict translational efficiency, estimate proteome composition, and evaluate tRNA abundance as a source of genetic variation. In this work, we utilized ribosome profiling, tRNAseq, and RNAseq and performed an integrative analysis in bovine tissues (kidney, liver and muscle) as well as murine myoblast cell lines (C2C12). By applying these methods to different bovine tissues, we were able to explore the interplay between tRNA availability and translational stalling events. Moreover, we have identified translationally regulated genes underlying tissue-specific biological processes and found that many upregulated and downregulated genes coincided with high and low translational efficiency respectively. We have also successfully defined stalling sites that depict the regulatory information encoded within the coding sequence of transcripts, which could control translation rate and facilitate proper protein folding. Through the implementation of these next generation sequencing (NGS) methods to cell culture, we were able to evaluate the translatome at various time points (0-min, 30-min, 60-min, and 4-h) post-induction of differentiation. Where most studies have focused on molecular changes between differentiating myoblasts and multinucleated myotubes that are present 7 d after differentiation, we focus on the earliest stages of muscle differentiation that initiate muscle development and therefore kickstart the process of meat production. This work offers an atlas of distinctive stalling sites across bovine tissues and various stages of muscle differentiation, which provides an opportunity to predict codon optimality and understand tissue-specific or time-specific mechanisms of regulating protein synthesis.

99 Noncoding Rnas Alter Our Interpretations of Genome to Phenome

Abstract Gene transcription and protein translation are core components in the process of gene expression. Previous research on the regulation of gene expression largely focuses on events prior to translation, including epigenetic regulation, transcription, and RNA processing. However, translation acts as an additional layer of regulation that plays an important role in gene expression and function. Highly expressed genes are thought to be codon-biased to support efficient translation, in which the encoded codons correspond to highly abundant tRNAs. Further, synonymous SNPs were once considered to be silent due to the degeneracy of the genetic code. However, synonymous SNPs may disturb protein abundance and function through alterations in translational efficiency and suboptimal pairing to lowly abundant tRNAs. Our previous study identified variation in tRNA expression within bovine liver and muscle tissue, suggesting tissue-specific modulation of translation. In addition, advances in sequencing technologies have recently permitted the study of the translatome, which refers to the entire population of mRNA associated with ribosomes for protein synthesis and can be investigated through ribosome profiling. Here, we applied Quantitative Mature tRNA sequencing (QuantM-tRNA-seq) and ribosome profiling to investigate the relationship between tRNA expression and translational stalling events. Moreover, we have identified translationally regulated genes underlying tissue-specific biological processes and found that many upregulated and downregulated genes coincided with high and low translational efficiency respectively. We have also successfully defined stalling sites that depict the regulatory information encoded within the coding sequence of transcripts, which could control translation rate and facilitate proper protein folding. This work offers an atlas of distinctive stalling sites across bovine tissues, which provides an opportunity to predict codon optimality and understand tissue-specific mechanisms of regulating protein synthesis.

High throughput detection of capillary stalling events with Bessel beam two-photon microscopy.

Brief disruptions in capillary flow, commonly referred to as capillary "stalling," have gained interest recently for their potential role in disrupting cerebral blood flow and oxygen delivery. Approaches to studying this phenomenon have been hindered by limited volumetric imaging rates and cumbersome manual analysis. The ability to precisely and efficiently quantify the dynamics of these events will be key in understanding their potential role in stroke and neurodegenerative diseases, such as Alzheimer's disease. Our study aimed to demonstrate that the fast volumetric imaging rates offered by Bessel beam two-photon microscopy combined with improved data analysis throughput allows for faster and more precise measurement of capillary stall dynamics. We found that while our analysis approach was unable to achieve full automation, we were able to cut analysis time in half while also finding stalling events that were missed in traditional blind manual analysis. The resulting data showed that our Bessel beam system was captured more stalling events compared to optical coherence tomography, particularly shorter stalling events. We then compare differences in stall dynamics between a young and old group of mice as well as a demonstrate changes in stalling before and after photothrombotic model of stroke. Finally, we also demonstrate the ability to monitor arteriole dynamics alongside stall dynamics. Bessel beam two-photon microscopy combined with high throughput analysis is a powerful tool for studying capillary stalling due to its ability to monitor hundreds of capillaries simultaneously at high frame rates.

A distinct mammalian disome collision interface harbors K63-linked polyubiquitination of uS10 to trigger hRQT-mediated subunit dissociation

Translational stalling events that result in ribosome collisions induce Ribosome-associated Quality Control (RQC) in order to degrade potentially toxic truncated nascent proteins. For RQC induction, the collided ribosomes are first marked by the Hel2/ZNF598 E3 ubiquitin ligase to recruit the RQT complex for subunit dissociation. In yeast, uS10 is polyubiquitinated by Hel2, whereas eS10 is preferentially monoubiquitinated by ZNF598 in human cells for an unknown reason. Here, we characterize the ubiquitination activity of ZNF598 and its importance for human RQT-mediated subunit dissociation using the endogenous XBP1u and poly(A) translation stallers. Cryo-EM analysis of a human collided disome reveals a distinct composite interface, with substantial differences to yeast collided disomes. Biochemical analysis of collided ribosomes shows that ZNF598 forms K63-linked polyubiquitin chains on uS10, which are decisive for mammalian RQC initiation. The human RQT (hRQT) complex composed only of ASCC3, ASCC2 and TRIP4 dissociates collided ribosomes dependent on the ATPase activity of ASCC3 and the ubiquitin-binding capacity of ASCC2. The hRQT-mediated subunit dissociation requires the K63-linked polyubiquitination of uS10, while monoubiquitination of eS10 or uS10 is not sufficient. Therefore, we conclude that ZNF598 functionally marks collided mammalian ribosomes by K63-linked polyubiquitination of uS10 for the trimeric hRQT complex-mediated subunit dissociation.

Under-Replicated DNA: The Byproduct of Large Genomes?

Simple SummaryHigher eukaryotic cells frequently enter mitosis with a certain load of under-replicated DNA, also referred to as unreplicated DNA, due to incomplete genomic DNA replication during the previous S phase. Double replication fork stalling events, when two converging forks irreversibly stall with no replication origin in between them, seem to be one of the major drivers of incomplete genomic replication in S phase. Genome stability is yet maintained in the vast majority of cells implying that cells must possess dedicated post-replicative mechanisms that allow for faithful repair of these seemingly unavoidable errors. Here, we provide a comprehensive overview of the mechanisms or events that cause, regulate and repair under-replicated DNA in eukaryotic cells.In this review, we provide an overview of how proliferating eukaryotic cells overcome one of the main threats to genome stability: incomplete genomic DNA replication during S phase. We discuss why it is currently accepted that double fork stalling (DFS) events are unavoidable events in higher eukaryotes with large genomes and which responses have evolved to cope with its main consequence: the presence of under-replicated DNA (UR-DNA) outside S phase. Particular emphasis is placed on the processes that constrain the detrimental effects of UR-DNA. We discuss how mitotic DNA synthesis (MiDAS), mitotic end joining events and 53BP1 nuclear bodies (53BP1-NBs) deal with such specific S phase DNA replication remnants during the subsequent phases of the cell cycle.

PMS

Single-source HTTP adaptive streaming solutions (HAS) have become the de facto solutions to deliver live video over the Internet. By avoiding video stalling events that are mainly caused by the lack of throughput at client or at server side, HAS solutions increase the end users’ quality of experience (QoE). We propose to pragmatically extend HAS with our MS-Stream solution that simultaneously utilizes several servers. MS-Stream aims at offering high QoE for live content delivery by exploiting expanded bandwidth and link diversity in distributed heterogeneous infrastructures. By leveraging end users’ connectivity capacities, we further extend the QoE and scalability capabilities of our proposal by exposing a hybrid P2P/multisource live-streaming solution (P2P/MS-Stream (PMS)), achieving trade-offs between the system’s scale and the end users’ QoE. We propose a distributed quality adaptation algorithm run by every peer, along with a local optimization method of the usage of the server infrastructure made available. Large-scale evaluations conducted with 300 peers located in France permits validating our approach and algorithms over flash crowd events and allow us to conclude that PMS can reach the optimal trade-offs between QoE and system scale.

ROSE: A Deep Learning Based Framework for Predicting Ribosome Stalling

We present a deep learning based framework, called ROSE, to accurately predict ribosome stalling events in translation elongation from coding sequences based on high‐throughput ribosome profiling data. Our validation results demonstrate the superior performance of ROSE over conventional prediction models. ROSE provides an effective index to estimate the likelihood of translational pausing at codon resolution and understand diverse putative regulatory factors of ribosome stalling. Also, the ribosome stalling landscape computed by ROSE can recover the functional interplay between ribosome stalling and cotranslational events in protein biogenesis, including protein targeting by the signal recognition particle (SRP) and protein secondary structure formation.

The polypeptide exit tunnel of the ribosomal large subunit requires assembly factors for proper construction and function

The final product of ribosome assembly consists of two ribosomal subunits capable of functioning with high fidelity. In eukaryotes, this process is facilitated by protein assembly factors that can promote proper formation of functional centers through structural probing, functional test‐driving, acting as a scaffold, or protecting the site from premature activity. The polypeptide exit tunnel (PET) of the ribosomal large subunit (LSU) is the conduit through which nascent polypeptide chains are passed as they are synthesized. Protein and RNA components of the PET lumen actively participate in translation by interacting with the nascent chain in order to regulate ribosome stalling events, thus regulating translation speed and gene expression. Recent cryo‐EM structures of immature LSU particles from different stages of assembly spanning the nucleoplasm to the cytoplasm have shown that the PET is sequentially probed by the C‐termini of three assembly factors: first, Nog1, then Rei1, and finally Reh1. The C‐termini of these three assembly factors mimic nascent chains, plugging the PET for almost the entirety of LSU assembly. No biological function, however, has been assigned to this redundant occupation of the PET. Here, we show that truncating PET‐inclusive residues from the C‐terminus of Nog1 causes LSU assembly defects that are exacerbated by analogous truncations to one or both of the Rei1 and Reh1 C‐termini. These defects manifest in processing of late immature ribosomal RNA intermediates. Interestingly, these assembly defects are further exacerbated at lower temperatures. Despite these mutants displaying slow growth at permissive temperatures, however, mature ribosomes are produced that may be defective in translation. Our data supports the model that Nog1 is necessary for efficient construction of the PET during early stages of assembly while Rei1 and Reh1 carry out checkpoints during later stages.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Quality-of-Experience Index for Streaming Video

With the rapid growth of streaming media applications, there has been a strong demand of quality-of-experience (QoE) measurement and QoE-driven video delivery technologies. Most existing methods rely on bitrate and global statistics of stalling events for QoE prediction. This is problematic for two reasons. First, using the same bitrate to encode different video content results in drastically different presentation quality. Second, the interactions between video presentation quality and playback stalling experiences are not accounted for. In this work, we first build a streaming video database and carry out a subjective user study to investigate the human responses to the combined effect of video compression, initial buffering, and stalling. We then propose a novel QoE prediction approach named Streaming QoE Index that accounts for the instantaneous quality degradation due to perceptual video presentation impairment, the playback stalling events, and the instantaneous interactions between them. Experimental results show that the proposed model is in close agreement with subjective opinions and significantly outperforms existing QoE models. The proposed model provides a highly effective and efficient meanings for QoE prediction in video streaming services. 1

Joker de Bruijn: Sequence Libraries to Cover All k-mers Using Joker Characters.

We present a deep learning based framework, called ROSE, to accurately predict ribosome stalling events in translation elongation from coding sequences based on high-throughput ribosome profiling data. Our validation results demonstrate the superior performance of ROSE over conventional prediction models. ROSE provides an effective index to estimate the likelihood of translational pausing at codon resolution and understand diverse putative regulatory factors of ribosome stalling. Also, the ribosome stalling landscape computed by ROSE can recover the functional interplay between ribosome stalling and cotranslational events in protein biogenesis, including protein targeting by the signal recognition particle (SRP) and protein secondary structure formation.

Targeted Detection of G‐Quadruplexes in Cellular RNAs

AbstractThe G‐quadruplex (G4) is a non‐canonical nucleic acid structure which regulates important cellular processes. RNA G4s have recently been shown to exist in human cells and be biologically significant. Described herein is a new approach to detect and map RNA G4s in cellular transcripts. This method exploits the specific control of RNA G4–cation and RNA G4–ligand interactions during reverse transcription, by using a selective reverse transcriptase to monitor RNA G4‐mediated reverse transcriptase stalling (RTS) events. Importantly, a ligation‐amplification strategy is coupled with RTS, and enables detection and mapping of G4s in important, low‐abundance cellular RNAs. Strong evidence is provided for G4 formation in full‐length cellular human telomerase RNA, offering important insights into its cellular function.

Targeted Detection of G-Quadruplexes in Cellular RNAs.

The G-quadruplex (G4) is a non-canonical nucleic acid structure which regulates important cellular processes. RNA G4s have recently been shown to exist in human cells and be biologically significant. Described herein is a new approach to detect and map RNA G4s in cellular transcripts. This method exploits the specific control of RNA G4–cation and RNA G4–ligand interactions during reverse transcription, by using a selective reverse transcriptase to monitor RNA G4-mediated reverse transcriptase stalling (RTS) events. Importantly, a ligation-amplification strategy is coupled with RTS, and enables detection and mapping of G4s in important, low-abundance cellular RNAs. Strong evidence is provided for G4 formation in full-length cellular human telomerase RNA, offering important insights into its cellular function.

YouSlow

Adaptive bitrate (ABR) technologies are being widely used in today's popular HTTP-based video streaming such as YouTube and Netflix. Such a rate-switching algorithm embedded in a video player is designed to improve video quality-of-experience (QoE) by selecting an appropriate resolution based on the analysis of network conditions while the video is playing. However, a bad viewing experience is often caused by the video player having difficulty estimating transit or client-side network conditions accurately. In order to analyze the ABR streaming performance, we developed YouSlow, a web browser plug-in that can detect and report live buffer stalling events to our analysis tool. Currently, YouSlow has collected more than 20,000 of YouTube video stalling events over 40 countries.

Propranolol Restricts the Mobility of Single EGF-Receptors on the Cell Surface before Their Internalization

The epidermal growth factor receptor is involved in morphogenesis, proliferation and cell migration. Its up-regulation during tumorigenesis makes this receptor an interesting therapeutic target. In the absence of the ligand, the inhibition of phosphatidic acid phosphohydrolase activity by propranolol treatment leads to internalization of empty/inactive receptors. The molecular events involved in this endocytosis remain unknown. Here, we quantified the effects of propranolol on the mobility of single quantum-dot labelled receptors before the actual internalization took place. The single receptors showed a clear stop-and-go motion; their diffusive tracks were continuously interrupted by sub-second stalling events, presumably caused by transient clustering. In the presence of propranolol we found that: i) the diffusion rate reduced by 22 %, which indicates an increase in drag of the receptor. Atomic force microscopy measurements did not show an increase of the effective membrane tension, such that clustering of the receptor remains the likely mechanism for its reduced mobility. ii) The receptor got frequently stalled for longer periods of multiple seconds, which may signal the first step of the internalization process.