Overlapping and distinct functions of CstF64 and CstF64τ in mammalian mRNA 3' processing

Abstract

mRNA 3' processing is dynamically regulated spatially and temporally. However, the underlying mechanisms remain poorly understood. CstF64τ is a paralog of the general mRNA 3' processing factor, CstF64, and has been implicated in mediating testis-specific mRNA alternative polyadenylation (APA). However, the functions of CstF64τ in mRNA 3' processing have not been systematically investigated. We carried out a comprehensive characterization of CstF64τ and compared its properties to those of CstF64. In contrast to previous reports, we found that both CstF64 and CstF64τ are widely expressed in mammalian tissues, and their protein levels display tissue-specific variations. We further demonstrated that CstF64 and CstF64τ have highly similar RNA-binding specificities both in vitro and in vivo. CstF64 and CstF64τ modulate one another's expression and play overlapping as well as distinct roles in regulating global APA profiles. Interestingly, protein interactome analyses revealed key differences between CstF64 and CstF64τ, including their interactions with another mRNA 3' processing factor, symplekin. Together, our study of CstF64 and CstF64τ revealed both functional overlap and specificity of these two important mRNA 3' processing factors and provided new insights into the regulatory mechanisms of mRNA 3' processing.

Keywords: 3′ end formation; alternative polyadenylation; mRNA processing; sequencing.

FIGURE 1.

Expression profiles of CstF-64 and CstF-64τ. (A) Dual color Western blot analysis of CstF64 and CstF64t expression level in different cell lines as marked. The red bands correspond to CstF64τ and the green bands correspond to CstF64. (B) Western blotting analyses of CstF64, CstF64τ, CstF77, CstF50, and GAPDH expression in the mouse tissues as marked. For each lane, ∼20 μg total protein was loaded. (*) This band migrated faster than CstF64 in other tissues and is a potential nonspecific band.

FIGURE 2.

Comparison of in vitro RNA binding specificity of CstF-64 and CstF-64τ using SELEX-seq. (A) A schematic representation of the SELEX-seq procedure (details described in the text and Materials and Methods). (B) Gel mobility shift assay using RNAs prepared from the random pool (library 1), CstF64-selected sequences (library 2), and CstF64τ-selected sequences (library 3), with GST-CstF64 (marked as CstF64) or GST-CstF64τ RRM (marked as CstF64τ). (C) Comparison of fold enrichment of all possible 10-mer RNA sequences in CstF64 and CstF64τ selected sequences: (x-axis) log10(frequency of each 10-mer sequence in library 2/its frequency in library 1); (y-axis) log10(frequency of each 10-mer sequence in library 3/its frequency in library 1). (D) Comparison of nucleotide composition of RNA sequences with fold enrichment in CstF64- and CstF64τ-selected sequences. Fold enrichment levels are marked on the bottom. The height of the boxes corresponds to the frequency of each nucleotide. (E) Quantification of the gel shift assays in Supplemental Figure S1. Three sequences were tested (different colored lines) with GST-CstF64-RRM (squares) or GST-CstF64τ-RRM (dots).

FIGURE 3.

Mapping CstF64τ-RNA interactions in vivo at the transcriptome level by iCLIP-seq analysis. (A) HeLa cells were UV irradiated or mock treated, and cell lysates were treated with RNase I as labeled. CstF64τ was IPed. After the 3′ linker ligation and 5′ end labeling, the purified RNP complexes were resolved by PAGE and visualized by phosphorimaging. CstF64τ and β-actin levels in all cell lysates were monitored by Western blotting. (B) Distribution of CstF64τ iCLIP tags in different regions compared to their frequency in the genome. (C) Distribution of the cleavage sites (green line), CstF64- (blue line), and CstF64τ binding sites (red line) relative to the closest upstream AWTAAA. Position 0 represents the 5′ end of AWTAAA. Weblogo of the top 20 over-represented motifs at CstF64τ crosslinking sites near poly(A) sites are shown in the inset. (D,E) CstF64 and CstF64τ iCLIP-seq results for Rps12 and Basp1 visualized on the UCSC genome browser.

FIGURE 4.

APA regulation by CstF64 and CstF64τ. (A) Western blotting analysis of control HeLa, CstF64-RNAi, and CstF64τ-RNAi cells. CstF64 and CstF64τ signals were quantified by using the ImageQuant program, and their levels in HeLa were set to 1. Relative levels in all samples are listed below. (B) Pairwise comparison of PAS usage in HeLa and CstF64τ-RNAi cells: (y-axis) log₁₀(proximal/distal)–HeLa; (x-axis) log₁₀(proximal/distal)–CstF64τ-RNAi. PAS pairs with statistically significant differences are highlighted in blue (DtoP shift) or red (PtoD shift). The numbers of PtoD and DtoP shifts are shown in the column graph in the inset. (C) UCSC Genome Browser tracks of the direct RNA sequencing results for CSTF3 in HeLa, CstF64-RNAi, and CstF64τ-RNAi cells. The proximal and distal PASs are marked on top. CstF64 and CstF64τ iCLIP data for the same region are also shown. (D) RT-qPCR validation of the APA changes in five selected genes: (y-axis) log2(extended 3′ UTR/common region)(CstF64τ-RNAi/HeLa). (E) A column graph showing the number of genes with significantly different APA profiles in CstF64-, CstF64τ-, or CstF64&τ-RNAi cells and the percentage of PtoD (red) and DtoP (blue) APA changes.

FIGURE 5.

Proteomic analyses of the CstF64 and CstF64τ interactomes. (A) Flag IPs were carried out using cell lysates from control HeLa cells or CstF64-3×Flag or CstF64τ-3×Flag cell lines. IP samples were resolved by SDS-PAGE and visualized by silver staining. (B) Western analyses of the IP samples described in A. (WB) Western blotting. (C) IP was carried out using anti-symplekin antibodies and HEK293 cell lysates. IP samples were resolved by SDS-PAGE and analyzed by Western blotting (WB).

FIGURE 6.

Characterization of the interactions between symplekin and CstF64/τ. (A) Symplekin and full-length CstF64/τ were prepared by in vitro translation and incubated in the specified combination. IP was carried out using control or anti-symplekin antibodies. The IP samples were resolved by SDS-PAGE and visualized by phosphorimaging: (*) an apparent truncated symplekin fragment. (B) GST pulldown assays with in vitro translated symplekin and GST or GST-CstF64-hinge or GST-CstF64τ-hinge. GST pulldown samples were resolved by SDS-PAGE and visualized by phosphorimaging. (C,D) IP experiments were carried out as described in A except that the P/G-rich domain (C) or the hinge-P/G-rich domain fragments (D) were used, respectively. (E) A summary of the in vitro binding assay results shown in A–D. (F) A schematic model for the interactions between symplekin and CstF64/τ. See text for details.