Genome level analysis of rice mRNA 3'-end processing signals and alternative polyadenylation

Abstract

The position of a poly(A) site of eukaryotic mRNA is determined by sequence signals in pre-mRNA and a group of polyadenylation factors. To reveal rice poly(A) signals at a genome level, we constructed a dataset of 55 742 authenticated poly(A) sites and characterized the poly(A) signals. This resulted in identifying the typical tripartite cis-elements, including FUE, NUE and CE, as previously observed in Arabidopsis. The average size of the 3'-UTR was 289 nucleotides. When mapped to the genome, however, 15% of these poly(A) sites were found to be located in the currently annotated intergenic regions. Moreover, an extensive alternative polyadenylation profile was evident where 50% of the genes analyzed had more than one unique poly(A) site (excluding microheterogeneity sites), and 13% had four or more poly(A) sites. About 4% of the analyzed genes possessed alternative poly(A) sites at their introns, 5'-UTRs, or protein coding regions. The authenticity of these alternative poly(A) sites was partially confirmed using MPSS data. Analysis of nucleotide profile and signal patterns indicated that there may be a different set of poly(A) signals for those poly(A) sites found in the coding regions. Based on the features of rice poly(A) signals, an updated algorithm termed PASS-Rice was designed to predict poly(A) sites.

Figure 1.

Single nucleotide profile comparison and the length of the 3′-UTRs of rice. (A) One nucleotide profile of the rice 3′-UTR and 100nt downstream of poly(A) sites. The regions of the poly(A) signals are shown. FUE, far upstream element; NUE, near upstream element; CE, cleavage element; CS, cleavage site or poly(A) site. The poly(A) site is at position -1. The upstream sequence (300 nt) of the poly(A) site is the minus designation, and downstream (100 nt) sequence is the plus designation. (B) One nucleotide profile of Arabidopsis 3′-UTR for comparison purposes. The arrangement is the same as in (A), and the dataset is as described (13). (C) Distribution of the 3′-UTR lengths in rice. Single sites, transcripts with only one poly(A) site found. APA, sites found in the 3′-UTR with more than one poly(A) site. Last sites, the furthest sites of the APA sites from stop codon. Total, based on all the 3′-UTR lengths. The average length of 289 nt is calculated from the total.

Figure 2.

Top-ranked hexamers in the rice poly(A) signal elements. (A) Hexamers from −35 to −10 in the NUE. (B) Hexamers from −10 to +15 in the CE. (C) Hexamers from −200 to −35 in the FUE. See Figure 1 legend for position annotation.

Figure 3.

An example (NUE) of how sequence logos were constructed. Dissimilarity distances between signals are calculated by using dynamic programming and then agglomerated by an R program. The suggested cutoff value of 2.6 was used. Hexamers in the same group were further aligned by ClustalW. The logos were generated using Web Logo tool based on ClustalW and their relative frequency in the derived region. The dotted lines indicate grouping regions.

Figure 4.

An example of APA of WRKY DNA binding domain-containing protein (LOC_Os01g47560) that is supported by rice MPSS data. The red and pink boxes represent exons and the 3′-UTR, respectively. Vertical arrows show the positions of poly(A) sites. Triangles in orange indicate MPSS signatures inside annotated gene/feature, and the triangle in purple indicates MPSS signatures between genes. The grey triangles are potential MPSS signatures, but not confirmed. The top panel (except for the arrows) was an output from the MPSS-rice web site. The numbers indicate 3 different transcripts resulting from the use of different poly(A) sites. A_(n) indicates a poly(A) tail. The vertical lines indicate splicing of the introns.

Figure 5.

Single nucleotide profiles and patterns around the APA site on the coding region. (A) One-nucleotide profiles of poly(A) sites in coding region. The location designation is the same as Figure 1. (B) Sequence logos in NUE region of poly(A) sites in coding region. Both TG- and AG-rich elements are unique.

Figure 6.

Representative outputs and evaluation parameters of PASS-Rice. (A) The Sn and Sp based on PASS-Rice. The Sp values were calculated based on rice intron, 5′-UTR, coding sequences (CDS) and a random sequence set generated by Markov chain (MC) based on the 2nd order trinucleotide distribution of rice 3′-UTR. (B) An example output of PASS-Rice using Los_03g61890 with multiple poly(A) sites. Triangles indicate the poly(A) sites confirmed by EST data.