PARalyzer: definition of RNA binding sites from PAR-CLIP short-read sequence data

Abstract

Crosslinking and immunoprecipitation (CLIP) protocols have made it possible to identify transcriptome-wide RNA-protein interaction sites. In particular, PAR-CLIP utilizes a photoactivatable nucleoside for more efficient crosslinking. We present an approach, centered on the novel PARalyzer tool, for mapping high-confidence sites from PAR-CLIP deep-sequencing data. We show that PARalyzer delineates sites with a high signal-to-noise ratio. Motif finding identifies the sequence preferences of RNA-binding proteins, as well as seed-matches for highly expressed microRNAs when profiling Argonaute proteins. Our study describes tailored analytical methods and provides guidelines for future efforts to utilize high-throughput sequencing in RNA biology. PARalyzer is available at http://www.genome.duke.edu/labs/ohler/research/PARalyzer/.

Figure 1

Example of PARalyzer interaction site identification. The entire genomic region corresponds to a single read-group from the Pumilio2 library. The orange region represents the nucleotides where the signal kernel density estimate is above background. The light pink locations are the full interaction sites extended by up to 5 nucleotides. A light gold box highlights the sequences that match the known Pumilio2 binding motif.

Figure 2

Nucleotide composition and RNA crosslinking likelihood centered on AGO1-4, QKI, PUM2, and IGF2BP1 interaction sites. The interaction site analysis is from all of the datasets: Quaking (QKI), Pumilio2 (PUM2), Insulin-like growth factor 2 binding protein 1 (IGF2BP1), and Argonaute 1 to 4 (AGO1 to -4). Heatmap: nucleotide composition, relative to a uniform background, of each individual binding site found in the respective genic regions. Barplot: likelihood of a T = > C conversion given that there is a 'T' at the given position. Unlike the heatmap, the barplot is not normalized by the number of reads mapping to an individual binding site. The red dotted line indicates the background conversion probability for all 'T's within the respective genic regions for each respective dataset. (a) Non-redundant seed-matches in 3' UTRs for the top 20 expressed miRNAs in the Argonaute dataset. 8 mer-m1 is a seed-match between the mRNA and nucleotides 1 to 8 of the miRNA seed sequence, 8 mer-A1 matches nucleotides 2 to 8 of the seed sequence paired with an A at position 1. 7 mer-1 m and 7 mer-A1 are similarly defined for nucleotides 1 to 7; 7 mer-m8 is a match utilizing nucleotides 2 to 8 of the seed sequence. 6 mer2-7 is a match utilizing nucleotides 2 to 7 of the seed sequence, and 6 mer3-8 utilizes nucleotides 3 to 8 of the sequence. (b) Motif matches for the two Quaking motifs in 3' UTRs, 5' UTRs, coding regions and introns. (c) Motif matches for the Pumilio 2 dataset in 3' UTRs, 5' UTRs, coding regions and introns. (d) Motif matches for the IGF2BP1 dataset in 3' UTRs, 5' UTRs, coding regions and introns.

Figure 3

Genomic location of PARalyzer generated interaction sites for four RNA-binding proteins. Locations of interaction sites that contained at least two T = > C conversions were compared to transcript sequences as annotated in ENSEMBL (release 57) [42]. The different repeat region classes were identified by RepeatMasker [44]. The following repeat types were collected for this analysis: low complexity repeat family (low complexity), long interspersed nuclear elements (LINE), short interspersed nuclear elements (SINE), DNA transposons (DNA), RNA repeat families (RNA), satellite repeat family (Satellite), rolling circle (RC), unknown repeat family (Unknown), long terminal repeats (LTR) and other repeats (Other).

Figure 4

Properties of Argonaute interaction site generation and their comparison to crosslink-centered regions. (a) Distribution of interaction site sizes for the Argonaute dataset for sites that fall within 3' UTRs and contain two or more T = > C conversion locations. The vertical red line represents the 41-nucleotide size of the Hafner et al. [7] crosslink-centered regions (CCRs). (b) Distribution of interaction site locations across a normalized 3' UTR for all clusters that have two or more T = > C conversion locations. (c) The signal-to-noise for the top 20 expressed miRNAs in the Argonaute dataset for both PARalyzer generated interaction sites and the Hafner et al. [7] CCRs located in 3' UTRs. (d) Average log2 signal-to-noise ratio of window size 21 across all 361 miRNAs reported expressed in Hafner et al. in the order of their expression rank.