Quantification of stochastic noise of splicing and polyadenylation in Entamoeba histolytica

Abstract

Alternative splicing and polyadenylation were observed pervasively in eukaryotic messenger RNAs. These alternative isoforms could either be consequences of physiological regulation or stochastic noise of RNA processing. To quantify the extent of stochastic noise in splicing and polyadenylation, we analyzed the alternative usage of splicing and polyadenylation sites in Entamoeba histolytica using RNA-Seq. First, we identified a large number of rarely spliced alternative junctions and then showed that the occurrence of these alternative splicing events is correlated with splicing site sequence, occurrence of constitutive splicing events and messenger RNA abundance. Our results implied the majority of these alternative splicing events are likely to be stochastic error of splicing machineries, and we estimated the corresponding error rates. Second, we observed extensive microheterogeneity of polyadenylation cleavage sites, and the extent of such microheterogeneity is correlated with the occurrence of constitutive cleavage events, suggesting most of such microheterogeneity is likely to be stochastic. Overall, we only observed a small fraction of alternative splicing and polyadenylation isoforms that are unlikely to be solely stochastic, implying the functional relevance of alternative splicing and polyadenylation in E. histolytica is limited. Lastly, we revised the gene models and annotated their 3′UTR in AmoebaDB, providing valuable resources to the community.

Regulation of gene expression in the protozoan parasite Entamoeba invadens: identification of core promoter elements and promoters with stage-specific expression patterns

“…We also analyzed the 5’ UTR length in select genes with high mRNA expression (Fragments Per Kilobase of exon per Million mapped reads (FPKM) values > 25,000) since those will have better 5’ sequence coverage. Determination of the polyA addition site was done using a method adapted from Hon et al (2013). RNA-seq data from 14 libraries representing seven developmental timepoints (Ehrenkaufer et al, 2013) were merged and mapped to the E. invadens genome using bowtie (Langmead et al, 2009) with the parameters: -v 3 --trim3 14.…”

“…The RNA-Seq methodology was used to improve the accuracy of genome annotation and identify untranslated regions as was demonstrated in E. histolytica (Hon et al, 2013), Tetrahymena (Xiong et al, 2012) and Plasmodium (Otto et al, 2010). Additionally, transcriptome data can be analyzed by bioinformatic approaches to identify conserved regulatory motifs as previously reported in Giardia lamblia (Tolba et al, 2013), Trypanosoma brucei (Mao et al, 2009), Tetrahymena thermophila (Xiong et al, 2012) and E. histolytica (Zamorano et al, 2008).…”

Sequencing of lariat termini in S. cerevisiae reveals 5′ splice sites, branch points, and novel splicing events

“…Indeed, these novel splicing events revealed two 5 ′ splice site sequences and nine branch site sequences that have not been reported previously in budding yeast (Supplemental Table S4), indicating a broader specificity of the spliceosome than previously suspected. Because we generally could not detect conservation of these splice sites in closely related yeast species, we suspect that many of these inefficient splicing events reflect splicing noise that stems from off-target binding of the splicing machinery, binding that has no functional role (Melamud and Moult 2009;Pickrell et al 2010;Hon et al 2013;Kawashima et al 2014). However, we cannot rule out the possibility that these splicing events reflect S. cerevisiae-specific regulatory mechanisms, in which splicing becomes efficient under conditions that differ from those utilized in this study.…”

Alternative splicing and the evolution of phenotypic novelty

“…Despite the various examples of alternatively spliced proteins having distinct functional roles, it is likely that a large proportion, or indeed the majority, of alternative splicing isoforms do not result in functional proteins [78][79][80][81][82]. An analysis of eight large-scale human proteomics experimentscollectively interrogating over 100 tissues, cell lines and developmental states with tandem mass spectrometry-found only 0.4% of the detected peptides are only derived from alternatively spliced transcripts [83], although a more recent study found that three quarters of isoforms with exon-skipping events and medium abundance in human cells are engaged by the ribosome and are likely to be translated [84].…”