Formation of mRNA 3' ends in eukaryotes: mechanism, regulation, and interrelationships with other steps in mRNA synthesis

Abstract

Formation of mRNA 3' ends in eukaryotes requires the interaction of transacting factors with cis-acting signal elements on the RNA precursor by two distinct mechanisms, one for the cleavage of most replication-dependent histone transcripts and the other for cleavage and polyadenylation of the majority of eukaryotic mRNAs. Most of the basic factors have now been identified, as well as some of the key protein-protein and RNA-protein interactions. This processing can be regulated by changing the levels or activity of basic factors or by using activators and repressors, many of which are components of the splicing machinery. These regulatory mechanisms act during differentiation, progression through the cell cycle, or viral infections. Recent findings suggest that the association of cleavage/polyadenylation factors with the transcriptional complex via the carboxyl-terminal domain of the RNA polymerase II (Pol II) large subunit is the means by which the cell restricts polyadenylation to Pol II transcripts. The processing of 3' ends is also important for transcription termination downstream of cleavage sites and for assembly of an export-competent mRNA. The progress of the last few years points to a remarkable coordination and cooperativity in the steps leading to the appearance of translatable mRNA in the cytoplasm.

FIG. 1

Schematic representation of poly(A) signals in animals, the yeast S. cerevisiae, and plants. USE, auxiliary upstream enhancer; DSE, downstream element; EE, efficiency element; PE, positioning element; FUE, far-upstream element; NUE, near-upstream element; nt, nucleotides. Adapted from reference .

FIG. 2

mRNA precursors are processed at their 3′ ends in a two-step reaction. A primary transcript is cleaved endonucleolytically at the poly(A) site, and this is followed by the addition of adenylate residues to the 3′ end of the upstream fragment to form a poly(A) tail. The factors responsible for each step of the reaction in mammals and in the yeast S. cerevisiae are indicated on each side.

FIG. 3

Schematic diagram of the bovine and yeast poly(A) polymerases. The scale at the top indicates the size in amino acids. The hatched bar indicates the conserved regions. DID and D mark the locations of the three aspartate residues essential for nucleotidyltransferase catalytic activity. Gray bars in different shades in the bovine PAP indicate the C terminus of PAP I and PAP II generated from alternative splicing. NLS, nuclear localization signal; RD, regulatory domain involved in inhibition by U1A and in coupling of splicing and polyadenylation; SpD, specificity domains; C-RBS, carboxyl-terminal RNA-binding site.

FIG. 4

Schematic representation of the mammalian (A) and yeast (B) mRNA 3′-end-processing complexes. (A) The mammalian cleavage complex assembles through a cooperative binding of CPSF at the AAUAAA signal and CstF at the U- or GU-rich sequence. CPSF-160 directly interacts with CstF-77 and PAP. The arrangement of CF I_m and CF II_m is not known. After the cleavage step, CPSF and PAP remain bound to the cleaved RNA and elongate the poly(A) tail in the presence of PAB II. (B) CF IA, Hrp1, and CF II are sufficient for the cleavage step in yeast. Poly(A) tail synthesis requires the addition of Pap1, PF I and Pab1. The Pan2/Pan3 deadenylase helps to regulate the poly(A) tail length.

FIG. 4

Schematic representation of the mammalian (A) and yeast (B) mRNA 3′-end-processing complexes. (A) The mammalian cleavage complex assembles through a cooperative binding of CPSF at the AAUAAA signal and CstF at the U- or GU-rich sequence. CPSF-160 directly interacts with CstF-77 and PAP. The arrangement of CF I_m and CF II_m is not known. After the cleavage step, CPSF and PAP remain bound to the cleaved RNA and elongate the poly(A) tail in the presence of PAB II. (B) CF IA, Hrp1, and CF II are sufficient for the cleavage step in yeast. Poly(A) tail synthesis requires the addition of Pap1, PF I and Pab1. The Pan2/Pan3 deadenylase helps to regulate the poly(A) tail length.

FIG. 5

Factors involved in cleavage of the histone mRNA precursor. SLBP1/HBP, stem-loop binding protein 1/hairpin-binding factor; HLF, heat-labile factor. Reproduced with modifications from reference with permission.

FIG. 6

Interrelationship of transcription and mRNA 3′-end formation. See the text for details.

FIG. 7

Exon definition and processing lead to formation of an export-competent mRNP. CBP, cap-binding proteins; U1, U2, the U1 and U2 snRNPs; SR, members of the serine- and arginine-rich family of splicing factors; U2AF, U2 snRNP auxiliary factor; SF-U1A, snRNP-free U1A complex; CPC, cleavage/polyadenylation complex; PAB II, poly(A)-binding protein II. The identity of factors directly contacting splicing factors during the bridging of terminal exons is not certain. This figure is adapted from a similar one in the review by Berget (39).

FIG. 8

Calcitonin/CGRP intron enhancer complex. Adapted from reference .

FIG. 9

Types of alternative polyadenylation choices include multiple poly(A) sites in the 3′ untranslated region (3′UTR) (A), a choice in defining the end of an exon by a 5′ splice site (5′SS) or a poly(A) site (B), and a choice of two different 3′-terminal exons (C). Adapted from reference .

FIG. 10

(A) Organization of exons and introns in calcitonin/CGRP pre-mRNA and the structure of the primary mRNA products produced in thyroid cells or neurons. (B) Alternative processing choices in the immunoglobulin M (IgM) heavy-chain (μ) precursor. (C) Structure of the integrated proviral DNA of the HIV-1 retrovirus and the primary transcript. The transcription initiation site is indicated by the arrow. USE, upstream polyadenylation enhancer; LTR, long-terminal repeat sequence; TAR, TAT-binding site; MSD, major splice donor used by all spliced transcripts. The promoter proximal and promoter-distal poly(A) sites and alternative splice donor (◊) and acceptor (▵) are also indicated. For ease of representation, the elements are not in correct size scale with respect to each other.

FIG. 10

(A) Organization of exons and introns in calcitonin/CGRP pre-mRNA and the structure of the primary mRNA products produced in thyroid cells or neurons. (B) Alternative processing choices in the immunoglobulin M (IgM) heavy-chain (μ) precursor. (C) Structure of the integrated proviral DNA of the HIV-1 retrovirus and the primary transcript. The transcription initiation site is indicated by the arrow. USE, upstream polyadenylation enhancer; LTR, long-terminal repeat sequence; TAR, TAT-binding site; MSD, major splice donor used by all spliced transcripts. The promoter proximal and promoter-distal poly(A) sites and alternative splice donor (◊) and acceptor (▵) are also indicated. For ease of representation, the elements are not in correct size scale with respect to each other.