Knockout of circRNAs by base editing back-splice sites of circularized exons

Many circular RNAs (circRNAs) are produced from back-splicing of exons of precursor mRNAs and are generally co-expressed with cognate linear RNAs. Methods for circRNA-specific knockout are lacking, largely due to sequence overlaps between forms. Here, we use base editors (BEs) for circRNA depletion. By targeting splice sites involved in both back-splicing and canonical splicing, BEs can repress circular and linear RNAs. Targeting sites predominantly for circRNA biogenesis, BEs could efficiently repress the production of circular but not linear RNAs. As hundreds of exons are predominantly back-spliced to produce circRNAs, this provides an efficient method to deplete circRNAs for functional study.

Minigene splicing assays reveal new insights into exonic variants of the SLC12A3 gene in Gitelman Syndrome.

Gitelman syndrome (GS) is a kind of salt-losing tubular disease, most of which is caused by SLC12A3 gene variants, and missense variants account for the majority. Recently, the phenomenon of exon skipping, in which exonic variants disrupt normal pre-mRNA splicing, has been related to a variety of diseases. The purpose of this study was to identify the effect of previously presumed missense SLC12A3 variants on pre-mRNA splicing using bioinformatics tools and minigenes. The results revealed that, among ten candidate variants, six variants (c.602G>A, c.602G>T, c.1667C>T, c.1925G>A, c.2548G>C and c.2549G>C) led to complete or incomplete exon skipping by affecting exonic splicing regulatory elements and/or disturbing canonical splice sites. It is worth mentioning that this is the largest study on pre-mRNA splicing of SLC12A3 exonic variants. In addition, our study emphasizes the importance of detecting splicing function at the mRNA level in GS and indicates that minigene analysis is a valuable tool for splicing functional assays of variants in vitro.

The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes

Metabolic labeling is a widely used tool to investigate different aspects of pre-mRNA splicing and RNA turnover. The labeling technology takes advantage of native cellular machineries where a nucleotide analog is readily taken up and incorporated into nascent RNA. One such analog is 4-thiouridine (4sU). Previous studies demonstrated that the uptake of 4sU at elevated concentrations (>50μM) and extended exposure led to inhibition of rRNA synthesis and processing, presumably induced by changes in RNA secondary structure. Thus, it is possible that 4sU incorporation may also interfere with splicing efficiency. To test this hypothesis, we carried out splicing analyses of pre-mRNA substrates with varying levels of 4sU incorporation (0–100%). We demonstrate that increased incorporation of 4sU into pre-mRNAs decreased splicing efficiency. The overall impact of 4sU labeling on pre-mRNA splicing efficiency negatively correlates with the strength of splice site signals such as the 3’ and the 5’ splice sites. Introns with weaker splice sites are more affected by the presence of 4sU. We also show that transcription by T7 polymerase and pre-mRNA degradation kinetics were impacted at the highest levels of 4sU incorporation. Increased incorporation of 4sU caused elevated levels of abortive transcripts, and fully labeled pre-mRNA is more stable than its uridine-only counterpart. Cell culture experiments show that a small number of alternative splicing events were modestly, but statistically significantly influenced by metabolic labeling with 4sU at concentrations considered to be tolerable (40 μM). We conclude that at high 4sU incorporation rates small, but noticeable changes in pre-mRNA splicing can be detected when splice sites deviate from consensus. Given these potential 4sU artifacts, we suggest that appropriate controls for metabolic labeling experiments need to be included in future labeling experiments.

Spliceator: multi-species splice site prediction using convolutional neural networks

Background Ab initio prediction of splice sites is an essential step in eukaryotic genome annotation. Recent predictors have exploited Deep Learning algorithms and reliable gene structures from model organisms. However, Deep Learning methods for non-model organisms are lacking. Results We developed Spliceator to predict splice sites in a wide range of species, including model and non-model organisms. Spliceator uses a convolutional neural network and is trained on carefully validated data from over 100 organisms. We show that Spliceator achieves consistently high accuracy (89–92%) compared to existing methods on independent benchmarks from human, fish, fly, worm, plant and protist organisms. Conclusions Spliceator is a new Deep Learning method trained on high-quality data, which can be used to predict splice sites in diverse organisms, ranging from human to protists, with consistently high accuracy.

Six splice site variations, three of them novel, in the ABO gene occurring in nine individuals with ABO subtypes

Background Nucleotide mutations in the ABO gene may reduce the activity of glycosyltransferase, resulting in lower levels of A or B antigen expression in red blood cells. Six known splice sites have been identified according to the database of red cell immunogenetics and the blood group terminology of the International Society of Blood Transfusion. Here, we describe six distinct splice site variants in individuals with ABO subtypes. Methods The ABO phenotype was examined using a conventional serological method. A polymerase chain reaction sequence-based typing method was used to examine the whole coding sequence of the ABO gene. The ABO gene haplotypes were studied using allele-specific primer amplification or cloning technology. In silico analytic tools were used to assess the functional effect of splice site variations. Results Six distinct variants in the ABO gene splice sites were identified in nine individuals with ABO subtypes, including c.28 + 1_2delGT, c.28 + 5G > A, c.28 + 5G > C, c.155 + 5G > A, c.204-1G > A and c.374 + 5G > A. c.28 + 1_2delGT was detected in an Aw individual, while c.28 + 5G > A, c.28 + 5G > C, and c.204-1G > A were detected in Bel individuals. c.155 + 5G > A was detected in one B3 and two AB3 individuals, whereas c.374 + 5G > A was identified in two Ael individuals. Three novel splice site variants (c.28 + 1_2delGT, c.28 + 5G > A and c.28 + 5G > C) in the ABO gene were discovered, all of which resulted in low antigen expression. In silico analysis revealed that all variants had the potential to alter splice transcripts. Conclusions Three novel splice site variations in the ABO gene were identified in Chinese individuals, resulting in decreased A or B antigen expression and the formation of ABO subtypes.

Modulation of pre-mRNA structure by hnRNP proteins regulates alternative splicing of MALT1

Alternative splicing is controlled by differential binding of trans-acting RNA binding proteins (RBPs) to cis-regulatory elements in intronic and exonic pre-mRNA regions. How secondary structure in the pre-mRNA transcripts affects recognition by RBPs and determines alternative exon usage is poorly understood. The MALT1 paracaspase is a key component of signaling pathways that mediate innate and adaptive immune responses. Alternative splicing of MALT1 exon7 is critical for controlling optimal T cell activation. Here, we demonstrate that processing of the MALT1 pre-mRNA depends on RNA structural elements that shield the 5′ and 3′ splice sites of the alternatively spliced exon7. By combining biochemical analyses with chemical probing and NMR we show that the RBPs hnRNP U and hnRNP L bind competitively and with comparable affinities to identical stem-loop RNA structures flanking the 5′ and 3′ splice sites of MALT1 exon7. While hnRNP U stabilizes RNA stem-loop conformations that maintain exon7 skipping, hnRNP L unwinds these RNA elements to facilitate recruitment of the essential splicing factor U2AF2 to promote exon7 inclusion. Our data represent a paradigm for the control of splice site selection by differential RBP binding and modulation of pre-mRNA structure.

Insights of Non-canonical Splice-site Variants on RNA Splicing in Patients with Congenital Hypothyroidism

Context Congenital hypothyroidism (CH) is caused by mutations in the genes for thyroid hormone synthesis. In our previous investigation of CH patients, ~53% of patients had mutations in either coding exons or canonical splice-sites of causative genes. Non-canonical splice-sites variants in the intron were detected but their pathogenic significance was not known. Objective To evaluate non-canonical splice-site variants on pre-mRNA splicing of CH-causing genes. Methods Next-generation sequencing data of 55 CH cases in 47 families were analyzed to identify rare intron variants. The effects of variants on pre-mRNA splicing were investigated by minigene RNA-splicing assays. Results Four intron variants were found in 3 patients: SLC26A4 c.1544 + 9C>T and c.1707 + 94C>T in one patient, and SLC5A5 c.970-48G>C and c.1652-97A>C in two other patients. The c.1707 + 94C>T and c.970-48G>C caused exons 15 and 16 skipping, and exon 8 skipping, respectively. The remaining variants had no effect on RNA splicing. Furthermore, we analyzed 28 previously reported non-canonical splice-site variants (4 in TG and 24 in SLC26A4). Among them, 15 variants (~54%) resulted in aberrant splicing and 13 variants had no effect on RNA splicing. These data were compared with three variant-prediction programs (FATHMM-XF, FATHMM-MKL, and CADD). Among 32 variants, FATHMM-XF, FATHMM-MKL, and CADD correctly predicted 20 (63%), 17 (53%), and 26 (81%) variants, respectively. Conclusions Two novel deep intron mutations have been identified in SLC26A4 and SLC5A5, bringing the total number of solved families with disease-causing mutations to ~45% in our cohort. Approximately 46% (13/28) reported non-canonical splice-site mutations do not disrupt pre-mRNA splicing. CADD provides highest prediction accuracy of non-canonical splice-site variants.

Intronization enhances expression of S-protein and other transgenes challenged by cryptic splicing

The natural habitat of SARS-CoV-2 is the cytoplasm of a mammalian cell where it replicates its genome and expresses its proteins. While SARS-CoV-2 genes and hence its codons are presumably well optimized for mammalian protein translation, they have not been sequence optimized for nuclear expression. The cDNA of the Spike protein harbors over a hundred predicted splice sites and produces mostly aberrant mRNA transcripts when expressed in the nucleus. While different codon optimization strategies increase the proportion of full-length mRNA, they do not directly address the underlying splicing issue with commonly detected cryptic splicing events hindering the full expression potential. Similar splicing characteristics were also observed in other transgenes. By inserting multiple short introns throughout different transgenes, significant improvement in expression was achieved, including >7-fold increase for Spike transgene. Provision of a more natural genomic landscape offers a novel way to achieve multi-fold improvement in transgene expression.