IntEREst: intron-exon retention estimator

BackgroundIn-depth study of the intron retention levels of transcripts provide insights on the mechanisms regulating pre-mRNA splicing efficiency. Additionally, detailed analysis of retained introns can link these introns to post-transcriptional regulation or identify aberrant splicing events in human diseases.ResultsWe present IntEREst, Intron–Exon Retention Estimator, an R package that supports rigorous analysis of non-annotated intron retention events (in addition to the ones annotated by RefSeq or similar databases), and support intra-sample in addition to inter-sample comparisons. It accepts binary sequence alignment/map (.bam) files as input and determines genome-wide estimates of intron retention or exon-exon junction levels. Moreover, it includes functions for comparing subsets of user-defined introns (e.g. U12-type vs U2-type) and its plotting functions allow visualization of the distribution of the retention levels of the introns. Statistical methods are adapted from the DESeq2, edgeR and DEXSeq R packages to extract the significantly more or less retained introns. Analyses can be performed either sequentially (on single core) or in parallel (on multiple cores). We used IntEREst to investigate the U12- and U2-type intron retention in human and plant RNAseq dataset with defects in the U12-dependent spliceosome due to mutations in the ZRSR2 component of this spliceosome. Additionally, we compared the retained introns discovered by IntEREst with that of other methods and studies.ConclusionIntEREst is an R package for Intron retention and exon-exon junction levels analysis of RNA-seq data. Both the human and plant analyses show that the U12-type introns are retained at higher level compared to the U2-type introns already in the control samples, but the retention is exacerbated in patient or plant samples carrying a mutated ZRSR2 gene. Intron retention events caused by ZRSR2 mutation that we discovered using IntEREst (DESeq2 based function) show considerable overlap with the retained introns discovered by other methods (e.g. IRFinder and edgeR based function of IntEREst). Our results indicate that increase in both the number of biological replicates and the depth of sequencing library promote the discovery of retained introns, but the effect of library size gradually decreases with more than 35 million reads mapped to the introns.

[1]  A. R. Jonckheere,et al.  A DISTRIBUTION-FREE k-SAMPLE TEST AGAINST ORDERED ALTERNATIVES , 1954 .

[2]  Marie-France Sagot,et al.  Theme: Computational Biology and Bioinformatics Computational Sciences for Biology, Medicine and the Environment , 2012 .

[3]  M. Frilander,et al.  The abundance of the spliceosomal snRNPs is not limiting the splicing of U12-type introns. , 2006, RNA.

[4]  B. Frey,et al.  transcriptomes Widespread intron retention in mammals functionally tunes Material , 2014 .

[5]  S. Miyano,et al.  Aberrant splicing of U12-type introns is the hallmark of ZRSR2 mutant myelodysplastic syndrome , 2014, Nature Communications.

[6]  A. Leutenegger,et al.  Association of TALS Developmental Disorder with Defect in Minor Splicing Component U4atac snRNA , 2011, Science.

[7]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[8]  T. J. Terpstra,et al.  The asymptotic normality and consistency of kendall's test against trend, when ties are present in one ranking , 1952 .

[9]  D. Greco,et al.  Global analysis of the nuclear processing of transcripts with unspliced U12-type introns by the exosome , 2014, Nucleic acids research.

[10]  Ping Zhang,et al.  Genetic algorithm with logistic regression for prediction of progression to Alzheimer's disease , 2014, BMC Bioinformatics.

[11]  W. Huber,et al.  Detecting differential usage of exons from RNA-seq data , 2012, Genome research.

[12]  Albin Sandelin,et al.  spliceR: an R package for classification of alternative splicing and prediction of coding potential from RNA-seq data , 2014 .

[13]  L. Pachter,et al.  A dynamic intron retention program enriched in RNA processing genes regulates gene expression during terminal erythropoiesis , 2015, Nucleic acids research.

[14]  Daniele Merico,et al.  Compound heterozygous mutations in the noncoding RNU4ATAC cause Roifman Syndrome by disrupting minor intron splicing , 2015, Nature Communications.

[15]  Mark D. Robinson,et al.  edgeR: a Bioconductor package for differential expression analysis of digital gene expression data , 2009, Bioinform..

[16]  G. Barton,et al.  How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use? , 2015, RNA.

[17]  E. Wang,et al.  Analysis and design of RNA sequencing experiments for identifying isoform regulation , 2010, Nature Methods.

[18]  Yamile Marquez,et al.  Complexity of the Alternative Splicing Landscape in Plants[C][W][OPEN] , 2013, Plant Cell.

[19]  E. Eyras,et al.  IRFinder: assessing the impact of intron retention on mammalian gene expression , 2017, Genome Biology.

[20]  I. Bozzoni,et al.  Expression of two Xenopus laevis ribosomal protein genes in injected frog oocytes. A specific splicing block interferes with the L1 RNA maturation. , 1984, Journal of Molecular Biology.

[21]  M. Frilander,et al.  Minor spliceosome and disease. , 2017, Seminars in cell & developmental biology.

[22]  W. Barbazuk,et al.  Aberrant splicing in maize rough endosperm3 reveals a conserved role for U12 splicing in eukaryotic multicellular development , 2017, Proceedings of the National Academy of Sciences.

[23]  Gael P. Alamancos,et al.  Leveraging transcript quantification for fast computation of alternative splicing profiles , 2014, bioRxiv.

[24]  L. Pérez-Jurado,et al.  Defective minor spliceosome mRNA processing results in isolated familial growth hormone deficiency , 2014, EMBO molecular medicine.

[25]  R. Durbin,et al.  A computational scan for U12-dependent introns in the human genome sequence. , 2001, Nucleic acids research.

[26]  Abhijit A. Patel,et al.  The splicing of U12‐type introns can be a rate‐limiting step in gene expression , 2002, The EMBO journal.

[27]  M. Frilander,et al.  Regulation of gene expression through inefficient splicing of U12-type introns , 2014, RNA biology.

[28]  Sandya Liyanarachchi,et al.  Mutations in U4atac snRNA, a Component of the Minor Spliceosome, in the Developmental Disorder MOPD I , 2011, Science.

[29]  M. Frilander,et al.  The significant other: splicing by the minor spliceosome , 2012, Wiley interdisciplinary reviews. RNA.

[30]  Tyler S. Alioto,et al.  U12DB: a database of orthologous U12-type spliceosomal introns , 2006, Nucleic Acids Res..

[31]  Amy Y. M. Au,et al.  Intron retention in mRNA: No longer nonsense , 2016, BioEssays : news and reviews in molecular, cellular and developmental biology.

[32]  E. Makeyev,et al.  Emerging functions of alternative splicing coupled with nonsense-mediated decay. , 2014, Biochemical Society transactions.

[33]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[34]  J. Malek,et al.  Mutation in noncoding RNA RNU12 causes early onset cerebellar ataxia , 2017, Annals of neurology.

[35]  Davis J. McCarthy,et al.  Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation , 2012, Nucleic acids research.

[36]  Wolfgang Huber,et al.  Drift and conservation of differential exon usage across tissues in primate species , 2013, Proceedings of the National Academy of Sciences.