Transcriptome-wide analyses of CstF64-RNA interactions in global regulation of mRNA alternative polyadenylation

Abstract

Cleavage stimulation factor 64 kDa (CstF64) is an essential pre-mRNA 3' processing factor and an important regulator of alternative polyadenylation (APA). Here we characterized CstF64-RNA interactions in vivo at the transcriptome level and investigated the role of CstF64 in global APA regulation through individual nucleotide resolution UV crosslinking and immunoprecipitation sequencing and direct RNA sequencing analyses. We observed highly specific CstF64-RNA interactions at poly(A) sites (PASs), and we provide evidence that such interactions are widely variable in affinity and may be differentially required for PAS recognition. Depletion of CstF64 by RNAi has a relatively small effect on the global APA profile, but codepletion of the CstF64 paralog CstF64τ leads to greater APA changes, most of which are characterized by the increased relative use of distal PASs. Finally, we found that CstF64 binds to thousands of dormant intronic PASs that are suppressed, at least in part, by U1 small nuclear ribonucleoproteins. Taken together, our findings provide insight into the mechanisms of PAS recognition and identify CstF64 as an important global regulator of APA.

Fig. 1.

iCLIP-seq mapping of CstF64-RNA interactions. (A) iCLIP mapping results. The three replicates are labeled. (B) Reproducibility of CstF64 crosslinking sites. The x-axis is the minimum cDNA count threshold, and the y-axis is the corresponding percentage of crosslinking sites reproduced in at least two (blue) or three (red) replicates. (C) Reproducibility of nucleotide composition at crosslinking sites. Frequencies of all possible pentamers overlapping the crosslinking sites are compared among three replicates. R² values and the top pentamer for each comparison are shown. The numbers on the axis represent the percentage of crosslinking sites that overlap with a specific pentamer. (D) Enrichment of CstF64 binding sites in different genomic regions. The log2 ratios between the observed and expected frequencies of reads in specified genomic regions are shown. (E) Position of the closest upstream A(A/U)UAAA for all CstF64 crosslinking sites (blue line) or random sites (black). Position 0 on the x-axis represents the CstF64 crosslinking site. (F) Overrepresented motifs at all CstF64 crosslinking sites or AAUAAA⁺ and AAUAAA⁻ crosslinking sites.

Fig. 2.

Differential CstF64–RNA interactions at PASs. (A) Distribution of CSs (green line, based on PAS-seq data) and CstF64 binding sites (blue line, based on iCLIP-seq data) relative to the closest upstream A(A/U)UAAA. Position 0 represents the 5′ end of AAUAAA. (B) PAS-seq and CstF64 iCLIP-seq results for BASP1 and RPS11 genes. The red arrows indicate the same regions downstream of the CSs. The read counts for the PAS-seq and iCLIP-seq peaks shown are listed on the right. (C and D) Web logos of the top 20 most greatly enriched motifs in the 0- to 20-nt and 20- to 40-nt regions downstream of the CSs of CstF64 CLIP⁺ and CLIP⁻ PASs.

Fig. 3.

CstF64–RNA interactions and PAS recognition. (A) Gel mobility shift assays using GST-CstF64-RRM (0, 25, and 50 μM) and the 60-nt sequences downstream of the CSs of the listed genes. SVL RNA was used as a positive control. (B) In vitro cleavage/polyadenylation assays in the presence of GST-CstF64-RRM (0, 30, and 60 μM). SVL was used as a control.

Fig. 4.

CstF64-mediated global APA regulation. (A) Western blot analysis of control HeLa, CstF64-RNAi, and CstF64&τ-RNAi cells. (B) Pairwise comparison of PAS use in HeLa, CstF64-RNAi, and CstF64&τ-RNAi cells. The y-axis shows log₁₀(proximal/distal)–HeLa. The x-axis shows log₁₀(proximal/distal)–CstF64-RNAi (Left) or –CstF64&τ-RNAi (Right). PAS pairs with statistically significant differences in use are highlighted in blue (higher use of proximal PAS in RNAi cells) or red (higher use of distal PAS in RNAi cells). (C) qRT-PCR verification of the APA changes in six genes. The y-axis shows the log2 ratio of RNAi/HeLa (extended/common). (D) Total normalized iCLIP signals for proximal-to-distal shift (red) and distal-to-proximal shift (blue) PAS pairs (the same highlighted PAS pairs as in B).