Genome-Wide Analysis of Polyadenylation Events in Schmidtea mediterranea

Abstract

In eukaryotes, 3′ untranslated regions (UTRs) play important roles in regulating posttranscriptional gene expression. The 3′UTR is defined by regulated cleavage/polyadenylation of the pre-mRNA. The advent of next-generation sequencing technology has now enabled us to identify these events on a genome-wide scale. In this study, we used poly(A)-position profiling by sequencing (3P-Seq) to capture all poly(A) sites across the genome of the freshwater planarian, Schmidtea mediterranea, an ideal model system for exploring the process of regeneration and stem cell function. We identified the 3′UTRs for ∼14,000 transcripts and thus improved the existing gene annotations. We found 97 transcripts, which are polyadenylated within an internal exon, resulting in the shrinking of the ORF and loss of a predicted protein domain. Around 40% of the transcripts in planaria were alternatively polyadenylated (ApA), resulting either in an altered 3′UTR or a change in coding sequence. We identified specific ApA transcript isoforms that were subjected to miRNA mediated gene regulation using degradome sequencing. In this study, we also confirmed a tissue-specific expression pattern for alternate polyadenylated transcripts. The insights from this study highlight the potential role of ApA in regulating the gene expression essential for planarian regeneration.

Efficient depletion of ribosomal RNA for RNA sequencing in planarians

“…3c). Moreover, using publicly available planarian poly(A)-position profiling by sequencing (3P-Seq) libraries [35], which allow the identification of 3′-ends of polyadenylated RNAs, no polyadenylation sites were detected in 16S rRNA. Therefore, we speculate that upon folding of 16S rRNA stretches of A nucleotides become exposed and facilitate the interaction with oligo-dT beads during transcript poly(A) selection.…”

Useful Bicistronic Reporter System for Studying Poly(A) Site-Defining cis Elements and Regulation of Alternative Polyadenylation

“…In silico analysis of ever-increasing transcriptome databases enables bioinformatic identification of putative pA sites, APA sites, and cis -acting elements [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 ]. Functional characterization of these putative pA sites, APA sites and cis -acting elements as well as of the candidate trans -acting factors and extracellular stimuli that regulate pA site strength and APA underlines the need for development of in vivo polyadenylation assay methods.…”

“…It was long held that pA sites are specified primarily by a highly conserved AAUAAA hexamer signal 10–30 bp 5′ to the cleavage site and a more variable U/UG-rich element 15–30 bp 3′ of the cleavage site. However, in silico analysis of the pA tail-containing transcripts from human, mouse, freshwater planarian ( Schmidtea mediterrane ), and fruit fly ( Drosophila melanogaster ) actually reveals three types of pA sites: canonical AAUAAA sites (specified by an upstream AAUAAA hexamer and a downstream U-/UG-rich element; 40–49%, depending on the species and data set examined), non-canonical ones (defined by one of the single-base variants of AAUAAA hexamer and a downstream U-/UG-rich element; 25–40%), and AAUAAA-like hexamer-independent ones (with no recognizable AAUAAA-like hexamer and U-/UG-rich element; 13–25%) [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 ]. Which cis elements define the 13–25% AAUAAA-like hexamer-independent pA sites remains underexplored.…”

Comprehensive annotation and characterization of planarian tRNA and tRNA-derived fragments (tRFs)

“…These events are controlled by underlying gene regulatory networks that spatially and temporally control the expression of specific genes thus orchestrating a coordinated regeneration process (13,14). Planarians possess several gene regulatory programs that are critical for neoblast function and regeneration (10,13,(15)(16)(17)(18)(19). Small RNAs are one of the key regulators of gene expression.…”