Mammalian tissues defective in nonsense-mediated mRNA decay display highly aberrant splicing patterns

Background

Nonsense-mediated mRNA decay (NMD) affects the outcome of alternative splicing by degrading mRNA isoforms with premature termination codons. Splicing regulators constitute important NMD targets; however, the extent to which loss of NMD causes extensive deregulation of alternative splicing has not previously been assayed in a global, unbiased manner. Here, we combine mouse genetics and RNA-seq to provide the first in vivo analysis of the global impact of NMD on splicing patterns in two primary mouse tissues ablated for the NMD factor UPF2.

Results

We developed a bioinformatic pipeline that maps RNA-seq data to a combinatorial exon database, predicts NMD-susceptibility for mRNA isoforms and calculates the distribution of major splice isoform classes. We present a catalog of NMD-regulated alternative splicing events, showing that isoforms of 30% of all expressed genes are upregulated in NMD-deficient cells and that NMD targets all major splicing classes. Importantly, NMD-dependent effects are not restricted to premature termination codon+ isoforms but also involve an abundance of splicing events that do not generate premature termination codons. Supporting their functional importance, the latter events are associated with high intronic conservation.

Conclusions

Our data demonstrate that NMD regulates alternative splicing outcomes through an intricate web of splicing regulators and that its loss leads to the deregulation of a panoply of splicing events, providing novel insights into its role in core- and tissue-specific regulation of gene expression. Thus, our study extends the importance of NMD from an mRNA quality pathway to a regulator of several layers of gene expression.     

Thioredoxin 1 as a serum marker for breast cancer and its use in combination with CEA or CA15-3 for improving the sensitivity of breast cancer diagnoses

Background

Influenza B and C are single-stranded RNA viruses that cause yearly epidemics and infections. Knowledge of RNA secondary structure generated by influenza B and C will be helpful in further understanding the role of RNA structure in the progression of influenza infection.

Findings

All available protein-coding sequences for influenza B and C were analyzed for regions with high potential for functional RNA secondary structure. On the basis of conserved RNA secondary structure with predicted high thermodynamic stability, putative structures were identified that contain splice sites in segment 8 of influenza B and segments 6 and 7 of influenza C. The sequence in segment 6 also contains three unused AUG start codon sites that are sequestered within a hairpin structure.

Conclusions

When added to previous studies on influenza A, the results suggest that influenza splicing may share common structural strategies for regulation of splicing. In particular, influenza 3′ splice sites are predicted to form secondary structures that can switch conformation to regulate splicing. Thus, these RNA structures present attractive targets for therapeutics aimed at targeting one or the other conformation.     

Evolutionarily emerged G tracts between the polypyrimidine tract and 3′ AG are splicing silencers enriched in genes involved in cancer

Background

The 3′ splice site (SS) at the end of pre-mRNA introns has a consensus sequence (Y)_nNYAG for constitutive splicing of mammalian genes. Deviation from this consensus could change or interrupt the usage of the splice site leading to alternative or aberrant splicing, which could affect normal cell function or even the development of diseases. We have shown that the position “N” can be replaced by a CA-rich RNA element called CaRRE1 to regulate the alternative splicing of a group of genes.

Results

Taking it a step further, we searched the human genome for purine-rich elements between the -3 and -10 positions of the 3′ splice sites of annotated introns. This identified several thousand such 3′SS; more than a thousand of them contain at least one copy of G tract. These sites deviate significantly from the consensus of constitutive splice sites and are highly associated with alterative splicing events, particularly alternative 3′ splice and intron retention. We show by mutagenesis analysis and RNA interference that the G tracts are splicing silencers and a group of the associated exons are controlled by the G tract binding proteins hnRNP H/F. Species comparison of a group of the 3′SS among vertebrates suggests that most (~87%) of the G tracts emerged in ancestors of mammals during evolution. Moreover, the host genes are most significantly associated with cancer.

Conclusion

We call these elements together with CaRRE1 regulatory RNA elements between the Py and 3′AG (REPA). The emergence of REPA in this highly constrained region indicates that this location has been remarkably permissive for the emergence of de novo regulatory RNA elements, even purine-rich motifs, in a large group of mammalian genes during evolution. This evolutionary change controls alternative splicing, likely to diversify proteomes for particular cellular functions.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1143) contains supplementary material, which is available to authorized users.     

A class of human exons with predicted distant branch points revealed by analysis of AG dinucleotide exclusion zones

Background

The three consensus elements at the 3' end of human introns - the branch point sequence, the polypyrimidine tract, and the 3' splice site AG dinucleotide - are usually closely spaced within the final 40 nucleotides of the intron. However, the branch point sequence and polypyrimidine tract of a few known alternatively spliced exons lie up to 400 nucleotides upstream of the 3' splice site. The extended regions between the distant branch points (dBPs) and their 3' splice site are marked by the absence of other AG dinucleotides. In many cases alternative splicing regulatory elements are located within this region.

Results

We have applied a simple algorithm, based on AG dinucleotide exclusion zones (AGEZ), to a large data set of verified human exons. We found a substantial number of exons with large AGEZs, which represent candidate dBP exons. We verified the importance of the predicted dBPs for splicing of some of these exons. This group of exons exhibits a higher than average prevalence of observed alternative splicing, and many of the exons are in genes with some human disease association.

Conclusion

The group of identified probable dBP exons are interesting first because they are likely to be alternatively spliced. Second, they are expected to be vulnerable to mutations within the entire extended AGEZ. Disruption of splicing of such exons, for example by mutations that lead to insertion of a new AG dinucleotide between the dBP and 3' splice site, could be readily understood even though the causative mutation might be remote from the conventional locations of splice site sequences.     

Analysis of splice donor and acceptor site function in a transposable gene trap derived from the maize element Activator

Gene trap vectors have been used in insertional mutagenesis in animal systems to clone genes with interesting patterns of expression. These vectors are designed to allow the expression of a reporter gene when the vector inserts into a transcribed region. In this paper we examine alternative splicing events that result in the expression of a GUS reporter gene carried on a Ds element which has been designed as a gene trap vector for plants. We have developed a rapid and reliable method based on PCR to study such events. Many splice donor sites were observed in the 3 Ac border. The relative frequency of utilisation of certain splice donor and acceptor sites differed between tobacco and Arabidopsis. A higher stringency of splicing was observed in Arabidopsis.     

Mutation analysis of the WNT4 gene in Han Chinese women with premature ovarian failure

Background

The WNT4 gene plays an important role in female sex determination and differentiation. It also contributes to maintaining of the ovaries and the survival of follicles.

Methods

We sequenced the coding region and splice sites of WNT4 in 145 Han Chinese women with premature ovarian failure (POF) and 200 healthy controls.

Results

Only one novel variation, in Exon 2 (195C > T), was detected among the women with POF. However, this synonymous variation did not result in a change in amino acid sequence (65 Asp > Asp). No further variants were found in any of the samples.

Conclusion

Although we cannot provide any evidence that it is a possible disease-causing gene, this study is the first attempt to investigate the possible role of WNT4 in Han Chinese women with POF.     

Analysis of the role of Caenorhabditis elegans GC-AG introns in regulated splicing

GC-AG introns represent 0.7% of total human pre-mRNA introns. To study the function of GC-AG introns in splicing regulation, 196 cDNA-confirmed GC-AG introns were identified in Caenorhabditis elegans. These represent 0.6% of the cDNA- confirmed intron data set for this organism. Eleven of these GC-AG introns are involved in alternative splicing. In a comparison of the genomic sequences of homologous genes between C.elegans and Caenorhabditis briggsae for 26 GC-AG introns, the C at the +2 position is conserved in only five of these introns. A system to experimentally test the function of GC-AG introns in alternative splicing was developed. Results from these experiments indicate that the conserved C at the +2 position of the tenth intron of the let-2 gene is essential for developmentally regulated alternative splicing. This C allows the splice donor to function as a very weak splice site that works in balance with an alternative GT splice donor. A weak GT splice donor can functionally replace the GC splice donor and allow for splicing regulation. These results indicate that while the majority of GC-AG introns appear to be constitutively spliced and have no evolutionary constraints to prevent them from being GT-AG introns, a subset of GC-AG introns is involved in alternative splicing and the C at the +2 position of these introns can have an important role in splicing regulation.     

Manipulating mRNA splicing by base editing in plants

Precursor-mRNAs(pre-mRNA) can be processed into one or more mature m RNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regulation, such as intronmediated enhancement(IME), whereas alternative splicing(AS) dramatically increases the protein diversity and gene functional regulation. However, the unavailability of mutants for individual spliced isoforms in plants has been a major limitation in studying the function of mRNA splicing. Here, we describe an efficient tool for manipulating the splicing of plant genes. Using a Cas9-directed base editor, we converted the 5′ splice sites in four Arabidopsis genes from the activated GT form to the inactive AT form. Silencing the AS of HAB 1.1(encoding a type 2 C phosphatase) validated its function in abscisic acid signaling, while perturbing the AS of RS31 A revealed its functional involvement in plant response to genotoxic treatment for the first time. Lastly,altering the constitutive splicing of Act2 via base editing facilitated the analysis of IME. This strategy provides an efficient tool for investigating the function and regulation of gene splicing in plants and other eukaryotes.