Functional Alternative Isoform Expression Analysis Using Long Read Technologies

RNA-seq has been claimed to be a powerful tool for the analysis of alternative splicing events. However, the analysis of the genome-wide functional implications of alternative isoforms expression remain a difficult task due to two main reasons: on one had the still existing limitations of short reads technologies and assembly algorithms to correctly predict full-length isoforms, and on the other hand, the lack of appropriate software to annotate and mine functional characteristics at the isoform resolution level.  In this work we present our initial results to develop a methodology for the functional analysis of alternative isoform expression. Our goal is to develop the methodology to address questions on the functional implications alternative isoform expression  (AIE) such as:

• Which fraction of a transcriptome is actually regulated by AIE?
• Which processes are specifically modulated by AIE ?
• To what extent AIE results in protein diversity or affects transcript post-transcriptional regulation?
• Are there functional relationships between different AIE events?
• Does AIE orchestrate relationships between coding and non-coding signals of transcript

To develop this methodology we are using as a benchmarking biological system mouse cell differentiation process from neural stem cells to oligodendrocytes. We have applied the smart-seq protocol [2] to obtain full-length RNA-seq libraries and sequenced them using PacBio and Illumina technologies. We obtained around 2 million PacBio subreads and 60 million Illumina reads per samples. The majority of the PacBio subreads contained both 3’ and 5’ sequencing primers and mapped at over 90% of the length of Refseq transcripts, indicating full-length transcript sequencing (Fig.1). PacBio reads were corrected and quantified with Illumina. We detected the expression of around 40,000 transcripts of which 16,000 transcripts were differentially expressed between the two cell types. An intensive functional annotation pipeline was applied on the transcript sequences to obtain rich functional labels at the isoform level: GO terms, Interpro domains, miRNA target sites, functional motifs at UTR regions, repetitive sequences and post-translational modifications.  Our preliminary results indicate notable functional annotation differences between the alternative isoforms of the same gene expressed in different cell types: for around 75% of the genes with AIE we could detect functional differences at either coding or non-coding aspects, being both type of functional differences of the same magnitude.  We have developed a number of functional profiling strategies to assess the cross-talk between functional elements of alternatively expressed isoforms. For example, we identified functional differences between genes regulated by gene differential expression and those regulated by alternative isoform expression. Similarly, we found up to 10 3’UTR transcript stability regulatory motifs that specifically associate to distinct neural differentiation functions. These results reveal the complexity of functional differences in alternative isoforms expression and set the way for the analysis of the genome-wide functional implications of alternative isoform expression.

 References

[1] Steijger et al. Nat Methods. 2013 Dec;10(12):1177-84

[2] Ramskold et al. Nat Biotechnol. 2012 Aug;30(8):777-82.