Abstract
The polyadenylation of RNA is a near-universal feature of RNA metabolism in eukaryotes. This process has been studied in the model alga Chlamydomonas reinhardtii using low-throughput (gene-by-gene) and high-throughput (transcriptome sequencing) approaches that recovered poly(A)-containing sequence tags which revealed interesting features of this critical process in Chlamydomonas. In this study, RNA polyadenylation has been studied using the so-called Poly(A) Tag Sequencing (PAT-Seq) approach. Specifically, PAT-Seq was used to study poly(A) site choice in cultures grown in four different media types—Tris-Phosphate (TP), Tris-Phosphate-Acetate (TAP), High-Salt (HS), and High-Salt-Acetate (HAS). The results indicate that: 1. As reported before, the motif UGUAA is the primary, and perhaps sole, cis-element that guides mRNA polyadenylation in the nucleus; 2. The scope of alternative polyadenylation events with the potential to change the coding sequences of mRNAs is limited; 3. Changes in poly(A) site choice in cultures grown in the different media types are very few in number and do not affect protein-coding potential; 4. Organellar polyadenylation is considerable and affects primarily ribosomal RNAs in the chloroplast and mitochondria; and 5. Organellar RNA polyadenylation is a dynamic process that is affected by the different media types used for cell growth.
Highlights
RNA processing in eukaryotic organisms is an intricate process comprised of multiple events that all appear to take place simultaneously before an mRNA is exported to the cytoplasm where translation to protein takes place [1,2,3]
Preparation and sequencing of poly(A) tag libraries prepared from Chlamydomonas
-called poly(A) tags (PATs), short cDNAs that contain the mRNA-poly(A) junction, were prepared from the isolated RNA following the protocol described in Ma et al and Pati et al [25, 26]
Summary
RNA processing in eukaryotic organisms is an intricate process comprised of multiple events that all appear to take place simultaneously before an mRNA is exported to the cytoplasm where translation to protein takes place [1,2,3]. The major events that comprise posttranscriptional RNA processing include 5’-end methylguanosine capping, intron/exon splicing, and 3’-end polyadenylation. Processing at the 3’-end of pre-mRNA transcripts has been well documented in animals, plants, and fungi where many similarities have been identified in addition to key differences amongst these kingdoms [4, 5]. PLOS ONE | DOI:10.1371/journal.pone.0146107 January 5, 2016
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