Means to an end: mechanisms of alternative polyadenylation of messenger RNA precursors

Abstract

Expression of mature messenger RNAs (mRNAs) requires appropriate transcription initiation and termination, as well as pre-mRNA processing by capping, splicing, cleavage, and polyadenylation. A core 3'-end processing complex carries out the cleavage and polyadenylation reactions, but many proteins have been implicated in the selection of polyadenylation sites among the multiple alternatives that eukaryotic genes typically have. In recent years, high-throughput approaches to map both the 3'-end processing sites as well as the binding sites of proteins that are involved in the selection of cleavage sites and in the processing reactions have been developed. Here, we review these approaches as well as the insights into the mechanisms of polyadenylation that emerged from genome-wide studies of polyadenylation across a range of cell types and states.

Figure 1

Composition of the human cleavage and polyadenylation complex. Different colors indicate individual protein subcomplexes. Components of the cleavage and polyadenylation specificity factor (CPSF) complex are depicted in close proximity to the cleavage and polyadenylation site, where CPSF1 recognizes the polyadenylation signal AAUAAA and CPSF3 is the endonuclease responsible for cleavage of the pre-messenger RNA (mRNA). CF I_m (cleavage factor) is depicted binding to UGUA motifs upstream of the cleavage site, while the cleavage stimulation factor (CstF) complex specifically interacts with a UG-rich region downstream of the cleavage site.

Figure 2

Outline of the main alternative polyadenylation (APA) patterns. One of the most studied patterns, tandem poly(A) sites, corresponds to multiple poly(A) sites being located in the 3′ untranslated region (UTR) of the terminal exon. Cleavage and polyadenylation at any of these sites will only lead to transcript isoforms that differ in the length of the 3′ UTR, but will not affect the protein-coding region of the messenger RNA (mRNA). Although referred to as an APA event, cleavage and polyadenylation at a different terminal exon is rather governed by alternative splicing decisions than APA. APA at cryptic poly(A) sites located in introns or exons can lead to truncated transcript isoforms with an altered coding sequence (CDS).

Figure 3

General outline of oligo(dT)-based 3′-end sequencing protocols (e.g., A-seq, PAS-seq, 3′-seq, and PolyA-seq17). Poly(A)⁺ RNA is usually isolated with oligo(dT)-coated beads, fragmented by alkaline hydrolysis, ribonuclease (RNase) treatment, or sonication, and oligo(dT)-adapter primers are used to reverse transcribe the RNA. Second-strand synthesis is accomplished with primers complementary to 5′ adapters, random hexamer-adapter primers, or by the Eberwine method (SMARTer kit by Clontech) where the reverse transcriptase (RT) adds a CCC tag to the cDNA that can be primed by an adapter-GGG molecule leading to a template switch. 5′ Adapter ligation can be omitted when the template switch method is used or second-strand synthesis after RT is performed with hexamer-5′ adapter primers (*). N is any nucleotide, B is any but A, and V is any but T.

Figure 4

Schematic outline of cross-linking and immunoprecipitation (CLIP) protocols for inferring protein interactions sites in RNAs. Many steps are interchangeable between protocols. Blotting after the sodium dodecyl sulfate (SDS) gel electrophoresis is frequently used to remove contaminating RNAs that are not cross-linked to proteins. Diagnostic mutations (substitutions, deletions, or insertions in all protocols, T → C mutations specifically in PAR-CLIP) are indicated.

Figure 5

Sequence composition around poly(A) sites. Poly(A) sites were determined based on publicly available 3′-end sequencing data (NCBI GEO entry GSE3019817), which we processed as described previously. (a) Position-dependent mononucleotide frequencies around the 10,000 poly(A) sites most frequently used in human cells. (b) Comparison of the frequency of occurrence of hexameric motifs at the same human poly(A) sites and their in vitro measured efficiency in polyadenylation.

Figure 6

Positional preferences of 3′-end processing subcomplexes. Profiles show the densities of T → C mutations (PAR-CLIP32) or reverse transcriptase (RT) truncation sites (iCLIP70) obtained in various cross-linking and immunoprecipitation (CLIP) experiments, relative to the 1000 most abundantly used 3′-end processing sites in the human genome.

Figure 7

Expression profiles of core and modulatory 3′-end processing factors in human tissues. The tissues are sorted from left to right in the order of increasing proliferation index (defined as in Ref 40). Expression data were obtained from BioGPS (http://biogps.org) and processed as described in the online Supporting Information. The numbers on the right side of each line represent the Spearman correlation coefficient between the expression levels of the indicated gene and the proliferative potential estimated from individual samples.

Figure 8

Evaluation of computational poly(A) site prediction tools. (a) Prediction of poly(A) sites: the 10,000 most frequently processed 3′ ends of human genes were used as the positive set and mononucleotide randomized variants of these sequences were used as the negative set to test the ability of POLYA_SVM, POLYAR, and Dragon PolyA spotter (DPS). Sequences and program outputs are available online as Supporting Information. (b) Prediction of relative use of tandem poly(A) sites in the human brain. We trained support vector classification models using either a string kernel on the nucleotide sequence at positions −40 to +40 around the poly(A) site or a RBF kernel using the poly(A) hexamer score and the G + U content in the 40 nt window downstream of the poly(A) site as input. Reported values are averaged accuracy values from a fourfold cross-validation.