Arg-tRNA synthetase links inflammatory metabolism to RNA splicing via nuclear condensates

SummaryCells respond to perturbations such as inflammation by sensing changes in metabolite levels. Especially prominent is arginine, which has long known connections to the inflammatory response. Here we show that depletion of arginine during inflammation decreased levels of arginyl-tRNA synthetase (ArgRS) in the nucleus. We found that nuclear ArgRS interacted with serine/arginine repetitive matrix protein 2 (SRRM2) in membrane-less, condensate-like, SRRM2-dependent nuclear speckles. This interaction impeded SRRM2 speckle trafficking and resulted in changes in alternative mRNA splicing. Splice site usage was regulated in opposite directions by ArgRS and SRRM2. These ArgRS- and SRRM2-dependent splicing changes cumulated in synthesis of different protein isoforms that altered cellular metabolism and peptide presentation to immune cells. Our findings delineate a novel mechanism whereby a tRNA synthetase responds to a metabolic change and modulates the splicing machinery via condensate trafficking for cellular responses to inflammatory injury.

HIV-1 sequences in lentiviral vector genomes can be substantially reduced without compromising transduction efficiency

Many lentiviral vectors used for gene therapy are derived from HIV-1. An optimal vector genome would include only the viral sequences required for transduction efficiency and gene expression to minimize the amount of foreign sequence inserted into a patient’s genome. However, it remains unclear whether all of the HIV-1 sequence in vector genomes is essential. To determine which viral sequences are required, we performed a systematic deletion analysis, which showed that most of the gag region and over 50% of the env region could be deleted. Because the splicing profile for lentiviral vectors is poorly characterized, we used long-read sequencing to determine canonical and cryptic splice site usage. Deleting specific regions of env sequence reduced the number of splicing events per transcript and increased the proportion of unspliced genomes. Finally, combining a large deletion in gag with repositioning the Rev-response element downstream of the 3’ R to prevent its reverse transcription showed that 1201 nucleotides of HIV-1 sequence can be removed from the integrated vector genome without substantially compromising transduction efficiency. Overall, this allows the creation of lentiviral vector genomes that contain minimal HIV-1 sequence, which could improve safety and transfer less viral sequence into a patient’s DNA.

Quantifying splice-site usage: a simple yet powerful approach to analyze splicing

RNA splicing, and variations in this process referred to as alternative splicing, are critical aspects of gene regulation in eukaryotes. From environmental responses in plants to being a primary link between genetic variation and disease in humans, splicing differences confer extensive phenotypic changes across diverse organisms (1–3). Regulation of splicing occurs through differential selection of splice sites in a splicing reaction, which results in variation in the abundance of isoforms and/or splicing events. However, genomic determinants that influence splice-site selection remain largely unknown. While traditional approaches for analyzing splicing rely on quantifying variant transcripts (i.e. isoforms) or splicing events (i.e. intron retention, exon skipping etc.) (4), recent approaches focus on analyzing complex/mutually exclusive splicing patterns (5–8). However, none of these approaches explicitly measure individual splice-site usage, which can provide valuable information about splice-site choice and its regulation. Here, we present a simple approach to quantify the empirical usage of individual splice sites reflecting their strength, which determines their selection in a splicing reaction. Splice-site strength/usage, as a quantitative phenotype, allows us to directly link genetic variation with usage of individual splice-sites. We demonstrate the power of this approach in defining the genomic determinants of splice-site choice through GWAS. Our pilot analysis with more than a thousand splice sites hints that sequence divergence in cis rather than trans is associated with variations in splicing among accessions of Arabidopsis thaliana. This approach allows deciphering principles of splicing and has broad implications from agriculture to medicine.

Exploration of the cyanidioschyzon merolae intron landscape

The eukaryotic process of pre-mRNA splicing involves the removal of noncoding intron sequences and the fusion of the remaining protein-coding exon sequences. The splicing reaction is catalyzed by the spliceosome, a dynamic multi-megadalton ribonucleoprotein complex that, in humans, is composed of 5 small nuclear RNAs (snRNAs) and over 200 associated proteins acting on more that 200,000 introns present within 25,000 genes. The unicellular red alga Cyanidioschyzon merolae possesses a more tractable splicing environment, with only 4 snRNAs and 75 associated proteins interacting with 27 annotated introns found in 26 our of 5,331 genes. Intron-rich genomes can confer benefits to their host species such as improved gene expression, incredible proteomic diversity, and increased genetic stability. This raises the question of why intron-poor C. merolae has retained such a small number of introns and a dramatically reduced spliceosome. A comprehensive investigation into the precise role that introns play in C. merolae would require the systematic removal of introns and an analysis of the effects thereof. The ability to elucidate the role of splicing in C. merolae via genome-wide intron deletion, however, hinges on the feasibility of establishing the efficiently scalable CRISPR genome engineering tool in C. merolae. It also follows that such an endeavour would require an accurate picture of the intron landscape of C. merolae, and since the number of annotated introns in C. merolae is relatively small, it is especially vital to determine whether any introns are missing from the C. merolae annotation. To that end, a stable and inducible Cas9-expressing strain of C. merolae was successfully developed. Transcriptome analysis using RNA-seq data revealed the discovery of 11 novel introns and 1 misannotated intron, as well as the presence of alternative splicing in the form of alternative splice site usage.

Nascent transcript folding plays a major role in determining RNA polymerase elongation rates

Transcription elongation rates are important for RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S.cerevisiae. Analysis of Miller chromatin spreads and mapping RNAPI using UV crosslinking, revealed a marked 5' bias and strikingly uneven local polymerase occupancy, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution; folding energy and G+C content. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high G+C within the transcription bubble slows elongation. We developed a mathematical model for RNAPI elongation, which confirmed the importance of nascent RNA folding in transcription. RNAPI from S.pombe was similarly sensitive to transcript folding, as were S.cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site usage indicating regulatory significance for transcript folding.

Global transcriptome analysis reveals circadian control of splicing events in Arabidopsis thaliana

SUMMARYThe circadian clock of Arabidopsis thaliana controls many physiological and molecular processes, allowing plants to anticipate daily changes in their environment. However, developing a detailed understanding of how oscillations in mRNA levels are connected to oscillations in post-transcriptional processes, such as splicing, has remained a challenge.Here we applied a combined approach using deep transcriptome sequencing and bioinformatics tools to identify novel circadian regulated genes and splicing events.Using a stringent approach, we identified 300 intron retention, 8 exon skipping, 79 alternative 3’ splice site usage, 48 alternative 5’ splice site usage, and 350 multiple (more than one event type) annotated events under circadian regulation. We also found 7 and 721 novel alternative exonic and intronic events. Depletion of the circadian regulated splicing factor AtSPF30 homolog, resulted in the disruption of a subset of clock controlled splicing events.Altogether, our global circadian RNA-seq coupled with an in silico, event centred, splicing analysis tool offers a new approach for studying the interplay between the circadian clock and the splicing machinery at a global scale. The identification of many circadian regulated splicing events broadens our current understanding of the level of control that the circadian clock has over this posttranscriptional regulatory layer.

Animal, fungi, and plant genome sequences harbour different non-canonical splice sites

Most protein encoding genes in eukaryotes contain introns which are interwoven with exons. After transcription, introns need to be removed in order to generate the final mRNA which can be translated into an amino acid sequence. Precise excision of introns by the spliceosome requires conserved dinucleotides which mark the splice sites. However, there are variations of the highly conserved combination of GT at the 5’ end and AG at the 3’ end of an intron in the genome. GC-AG and AT-AC are two major non-canonical splice site combinations which have been known for years. During the last years, various minor non-canonical splice site combinations were detected with numerous dinucleotide permutations. Here we expand systematic investigations of non-canonical splice site combinations in plants to all eukaryotes by analysing fungal and animal genome sequences. Comparisons of splice site combinations between these three kingdoms revealed several differences such as a substantially increased CT-AC frequency in fungal genome sequences. Canonical GT-AG splice site combinations in antisense transcripts could be one explanation for this observation. In addition, high numbers of GA-AG splice site combinations were observed in Eurytemora affinis and Oikopleura dioica. A variant in one U1 snRNA isoform might allow the recognition of GA as 5’ splice site. In depth investigation of splice site usage based on RNA-Seq read mappings indicates a generally higher flexibility of the 3’ splice site compared to the 5’ splice site across animals, fungi, and plants.