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.
Digital Object Identifier (DOI)
This research was supported by NSF Award MCB-1243849 and an REU Supplement to NSF Award IOS-0817818.
The Illumina sequences generated in this study are available in Bioproject PRJNA294481, accessed through the NCBI web site. These sequences have not been demultiplexed or otherwise processed; these manipulations may be performed following the procedures described in S1 File.
Bell, Stephen A.; Shen, Chi; Brown, Alishea; and Hunt, Arthur G., "Experimental Genome-Wide Determination of RNA Polyadenylation in Chlamydomonas reinhardtii" (2016). Plant and Soil Sciences Faculty Publications. 74.
S1 Fig. Growth stage analysis of Chlamydomonas cultures grown in the four different media types used in this study.
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S2 Fig. Principal component analysis of gene expression for the twelve Chlamydomonas PAT libraries created.
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S3 Fig. Plots of the position-by-position base composition of poly(A) sites that map cannot be linked with recognizable UGUAA motifs.
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S4 Fig. Characteristics of poly(A) sites that map to unannotated genomic positions.
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S5 Fig. Genome browser representations of genes whose poly(A) site usage varies in one or more of the experimental conditions used for this study.
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S6 Fig. Close-up genome browser illustrations of mappings to three chloroplast genes whose poly(A) sites have been reported elsewhere (see the text).
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S1 File. Computational pipeline used to analyze the sequencing data.
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S2 File. Summaries of read mapping outcomes for the twelve libraries.
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S3 File. Results of the gene expression analysis conducted using CLC Genomics Workbench.
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S4 File. Poly(A) site (PAS) list.
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S5 File. Poly(A) site Cluster (PAC) list.
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S6 File. Motif discovery for poly(A) sites that map to 5’-UTRs, introns, and protein-coding regions.
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S7 File. Annotation features closest to PACs that map to unannotated regions.
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S8 File. DEXSeq analysis output.
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S9 File. Lists of PAS for the Chlamydomonas organelles.
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S10 File. Results of the chloroplast gene expression analysis conducted using CLC Genomics Workbench.