R2Dtool: integration and visualization of isoform-resolved RNA features.

Motivation: Long-read RNA sequencing enables the mapping of RNA modifications, structures, and protein-interaction sites at the resolution of individual transcript isoforms. To understand the functions of these RNA features, it is critical to analyze them in the context of transcriptomic and genomic annotations, such as open reading frames and splice junctions.

Results: We have developed R2Dtool, a bioinformatics tool that integrates transcript-mapped information with transcript and genome annotations, allowing for the isoform-resolved analytics and graphical representation of RNA features in their genomic context.

Substrate diversity of NSUN enzymes and links of 5-methylcytosine to mRNA translation and turnover.

Maps of the RNA modification 5-methylcytosine (mC) often diverge markedly not only because of differences in detection methods, data depand analysis pipelines but also biological factors. We re-analysed bisulfite RNA sequencing datasets from five human cell lines and seven tissues using a coherent mC site calling pipeline. With the resulting union list of 6,393 mC sites, we studied site distribution, enzymology, interaction with RNA-binding proteins and molecular function.

Biochemical-free enrichment or depletion of RNA classes in real-time during direct RNA sequencing with RISER.

The heterogeneous composition of cellular transcriptomes poses a major challenge for detecting weakly expressed RNA classes, as they can be obscured by abundant RNAs. Although biochemical protocols can enrich or deplete specified RNAs, they are time-consuming, expensive and can compromise RNA integrity. Here we introduce RISER, a biochemical-free technology for the real-time enrichment or depletion of RNA classes.

Dynamics of mRNA fate during light stress and recovery: from transcription to stability and translation.

Transcript stability is an important determinant of its abundance and, consequently, translational output. Transcript destabilisation can be rapid and is well suited for modulating the cellular response. However, it is unclear the extent to which RNA stability is altered under changing environmental conditions in plants.

An Improved Method for Purification of the Residual Bodies from the Seminiferous Tubules of Mice.

Human fertility is declining in Western countries, and it is becoming increasingly clear that male infertility plays a pivotal role in the overall fertility decline. To understand the process that drives successful male germ cell maturation, the study of spermatogenesis of model organisms, such as mice, is essential. Residual bodies (RBs) play an important role in the last stages of spermatogenesis.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells.

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and 'damage control' during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods.

Translational control in cell ageing: an update.

Cellular ageing is one of the main drivers of organismal ageing and holds keys towards improving the longevity and quality of the extended life. Elucidating mechanisms underlying the emergence of the aged cells as well as their altered responses to the environment will help understanding the evolutionarily defined longevity preferences across species with different strategies of survival. Much is understood about the role of alterations in the DNA, including many epigenetic modifications such as methylation, in relation to the aged cell phenotype.

Translation complex stabilization on messenger RNA and footprint profiling to study the RNA responses and dynamics of protein biosynthesis in the cells.

During protein biosynthesis, ribosomes bind to messenger (m)RNA, locate its protein-coding information, and translate the nucleotide triplets sequentially as codons into the corresponding sequence of amino acids, forming proteins. Non-coding mRNA features, such as 5' and 3' untranslated regions (UTRs), start sites or stop codons of different efficiency, stretches of slower or faster code and nascent polypeptide interactions can alter the translation rates transcript-wise. Most of the homeostatic and signal response pathways of the cells converge on individual mRNA control, as well as alter the global translation output.

Selective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes.

Translational control targeting the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast translation complex profile sequencing (TCP-seq), related to ribosome profiling, and adapted it for mammalian cells.

Migration of Small Ribosomal Subunits on the 5' Untranslated Regions of Capped Messenger RNA.

Several control mechanisms of eukaryotic gene expression target the initiation step of mRNA translation. The canonical translation initiation pathway begins with cap-dependent attachment of the small ribosomal subunit (SSU) to the messenger ribonucleic acid (mRNA) followed by an energy-dependent, sequential 'scanning' of the 5' untranslated regions (UTRs). Scanning through the 5'UTR requires the adenosine triphosphate (ATP)-dependent RNA helicase eukaryotic initiation factor (eIF) 4A and its efficiency contributes to the specific rate of protein synthesis.

Control of Translation at the Initiation Phase During Glucose Starvation in Yeast.

Glucose is one of the most important sources of carbon across all life. Glucose starvation is a key stress relevant to all eukaryotic cells. Glucose starvation responses have important implications in diseases, such as diabetes and cancer.

Insights into the biogenesis and potential functions of exonic circular RNA.

Circular RNAs (circRNAs) exhibit unique properties due to their covalently closed nature. Models of circRNAs synthesis and function are emerging but much remains undefined about this surprisingly prevalent class of RNA. Here, we identified exonic circRNAs from human and mouse RNA-sequencing datasets, documenting multiple new examples.

Translation initiation by cap-dependent ribosome recruitment: Recent insights and open questions.

Gene expression universally relies on protein synthesis, where ribosomes recognize and decode the messenger RNA template by cycling through translation initiation, elongation, and termination phases. All aspects of translation have been studied for decades using the tools of biochemistry and molecular biology available at the time. Here, we focus on the mechanism of translation initiation in eukaryotes, which is remarkably more complex than prokaryotic initiation and is the target of multiple types of regulatory intervention.

Translation complex profile sequencing to study the in vivo dynamics of mRNA-ribosome interactions during translation initiation, elongation and termination.

Messenger RNA (mRNA) translation is a tightly controlled process that is integral to gene expression. It features intricate and dynamic interactions of the small and large subunits of the ribosome with mRNAs, aided by multiple auxiliary factors during distinct initiation, elongation and termination phases. The recently developed ribosome profiling method can generate transcriptome-wide surveys of translation and its regulation.

Dynamics of ribosome scanning and recycling revealed by translation complex profiling.

Regulation of messenger RNA translation is central to eukaryotic gene expression control. Regulatory inputs are specified by them RNA untranslated regions (UTRs) and often target translation initiation. Initiation involves binding of the 40S ribosomal small subunit (SSU) and associated eukaryotic initiation factors (eIFs)near the mRNA 5′ cap; the SSU then scans in the 3′ direction until it detects the start codon and is joined by the 60S ribosomal large subunit (LSU) to form the 80S ribosome.

Probe-Directed Degradation (PDD) for Flexible Removal of Unwanted cDNA Sequences from RNA-Seq Libraries.

Most applications for RNA-seq require the depletion of abundant transcripts to gain greater coverage of the underlying transcriptome. The sequences to be targeted for depletion depend on application and species and in many cases may not be supported by commercial depletion kits. This unit describes a method for generating RNA-seq libraries that incorporates probe-directed degradation (PDD), which can deplete any unwanted sequence set, with the low-bias split-adapter method of library generation (although many other library generation methods are in principle compatible).

Probing the closed-loop model of mRNA translation in living cells.

The mRNA closed-loop, formed through interactions between the cap structure, poly(A) tail, eIF4E, eIF4G and PAB, features centrally in models of eukaryotic translation initiation, although direct support for its existence in vivo is not well established. Here, we investigated the closed-loop using a combination of mRNP isolation from rapidly cross-linked cells and high-throughput qPCR. Using the interaction between these factors and the opposing ends of mRNAs as a proxy for the closed-loop, we provide evidence that it is prevalent for eIF4E/4G-bound but unexpectedly sparse for PAB1-bound mRNAs, suggesting it primarily occurs during a distinct phase of polysome assembly.

Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage.

Background: A major hurdle to transcriptome profiling by deep-sequencing technologies is that abundant transcripts, such as rRNAs, can overwhelm the libraries, severely reducing transcriptome-wide coverage. Methods for depletion of such unwanted sequences typically require treatment of RNA samples prior to library preparation, are costly and not suited to unusual species and applications. Here we describe Probe-Directed Degradation (PDD), an approach that employs hybridisation to DNA oligonucleotides at the single-stranded cDNA library stage and digestion with Duplex-Specific Nuclease (DSN).

Insight into the structural organization of the omega leader of TMV RNA: the role of various regions of the sequence in the formation of a compact structure of the omega RNA.

The 5'-untranslated sequence of tobacco mosaic virus RNA--the so called omega leader--is a well-known translational enhancer. The structure of the omega RNA has unusual features. Despite the absence of extensive secondary structure of the Watson-Crick type, the omega RNA possesses a stable compact conformation.

Quantitative analysis of ribosome-mRNA complexes at different translation stages.

Inhibition of primer extension by ribosome-mRNA complexes (toeprinting) is a proven and powerful technique for studying mechanisms of mRNA translation. Here we have assayed an advanced toeprinting approach that employs fluorescently labeled DNA primers, followed by capillary electrophoresis utilizing standard instruments for sequencing and fragment analysis. We demonstrate that this improved technique is not merely fast and cost-effective, but also brings the primer extension inhibition method up to the next level.

Poly(A) leader of eukaryotic mRNA bypasses the dependence of translation on initiation factors.

Eukaryotic mRNAs in which a poly(A) sequence precedes the initiation codon are known to exhibit a significantly enhanced cap-independent translation, both in vivo and in cell-free translation systems. Consistent with high expression levels of poxviral mRNAs, they contain poly(A) sequences at their 5' ends, immediately before the initiation AUG codon. Here we show that poly(A) as a leader sequence in mRNA constructs promotes the recruitment of the 40S ribosomal subunits and the efficient formation of initiation complexes at cognate AUG initiation codons in the absence of two essential translation initiation factors, eIF3 and eIF4F.

Dissociation of eIF1 from the 40S ribosomal subunit is a key step in start codon selection in vivo.

Selection of the AUG start codon is a key step in translation initiation requiring hydrolysis of GTP in the eIF2*GTP*Met-tRNA(i)(Met) ternary complex (TC) and subsequent P(i) release from eIF2*GDP*P(i). It is thought that eIF1 prevents recognition of non-AUGs by promoting scanning and blocking P(i) release at non-AUG codons. We show that Sui(-) mutations in Saccharomyces cerevisiae eIF1, which increase initiation at UUG codons, reduce interaction of eIF1 with 40S subunits in vitro and in vivo, and both defects are diminished in cells by overexpressing the mutant proteins.

The leader sequence of tobacco mosaic virus RNA devoid of Watson-Crick secondary structure possesses a cooperatively melted, compact conformation.

The 5'-untranslated region (5'-UTR) of RNA of tobacco mosaic virus (TMV), called omega sequence, is known as an mRNA leader promoting efficient initiation of translation. The central part of the sequence consists of many CAA repeats, which were reported to be mainly responsible for the enhancing activity of the omega leader. In this work we synthesized the polyribonucleotides containing either the natural omega sequence or the regular (CAA)(n) sequence, and studied them using UV spectrophotometry and analytical ultracentrifugation methods.