Roles of mRNA poly(A) tails in regulation of eukaryotic gene expression

Abstract

In eukaryotes, poly(A) tails are present on almost every mRNA. Early experiments led to the hypothesis that poly(A) tails and the cytoplasmic polyadenylate-binding protein (PABPC) promote translation and prevent mRNA degradation, but the details remained unclear. More recent data suggest that the role of poly(A) tails is much more complex: poly(A)-binding protein can stimulate poly(A) tail removal (deadenylation) and the poly(A) tails of stable, highly translated mRNAs at steady state are much shorter than expected. Furthermore, the rate of translation elongation affects deadenylation. Consequently, the interplay between poly(A) tails, PABPC, translation and mRNA decay has a major role in gene regulation. In this Review, we discuss recent work that is revolutionizing our understanding of the roles of poly(A) tails in the cytoplasm. Specifically, we discuss the roles of poly(A) tails in translation and control of mRNA stability and how poly(A) tails are removed by exonucleases (deadenylases), including CCR4-NOT and PAN2-PAN3. We also discuss how deadenylation rate is determined, the integration of deadenylation with other cellular processes and the function of PABPC. We conclude with an outlook for the future of research in this field.

Box 1 Figure

Structure of poly(A) RNA (purple) bound to the Pan2 exonuclease domain (pink) (PDB 6R9J). The poly(A) RNA forms an A-form like, single-stranded helix where the bases are stacked on top of each other.

Figure 1. mRNA Poly(A) tails function as master regulators of gene expression in the cytoplasm.

In the nucleus, pre-mRNAs are transcribed by RNA polymerase II (Pol II) and processed, including 5′-capping, splicing and 3′-cleavage and polyadenylation. Nuclear poly(A) binding proteins (PABPN) function in the nucleus to control poly(A) tail addition. Mature, polyadenylated mRNAs are exported into the cytoplasm. Cytoplasmic poly(A)- binding protein (PABPC) binds poly(A) and promotes translation by the 80S ribosome. Poly(A) tails and PABPC also influence mRNA stability: removal or shortening of the poly(A) tail (deadenylation) releases PABPC and leads to mRNA degradation.

Figure 2. mRNA poly(A) tails stimulate translation.

A Structure of eukaryotic poly(A)- binding protein (PABPC – Pab1 in yeast, PABPC1 in mammals). Domain diagram of the conserved PABPC is shown with the position of the four RNA-recognition motif (RRM) domains and the Mademoiselle (MLLE) domain indicated^,. Below, a crystal structure of RRM1-RRM2 (cartoon) of yeast Pab1 bound to poly(A) RNA (sticks) is shown (PDB 1CVJ). B Arrangement of PABPC on RNA. Two PABPC molecules can bind a 60 nt poly(A) tail. RRMs 1 and 2 have the highest affinity and specificity for poly(A) and require ~12 As for high affinity binding^,. Full-length PABPC footprints ~30 nt and adjacent PABPC molecules interact with each other^,,. RRM4 may bind to the 3′-UTR. C The mRNA 5′-cap (magenta circle) and 3′-poly(A) tail act synergistically to stimulate gene expression in eukaryotes. The relative amount of protein produced from reporter mRNAs with and without 5′ cap and poly(A) tail in plant, animal and yeast cells are depicted. D Closed-loop model. The eukaryotic translation initiation factor 4E (eIF4E) binds the 5′-cap. eIF4G binds both eIF4E and PABPC, as well as the RNA helicase eIF4A, and this is thought to stimulate recruitment of the small (40S) ribosomal subunit. 40S assembles with a large (60S) ribosomal subunit on a start codon to form a translation-competent 80S ribosome.

Figure 3. Eukaryotic mRNA deadenylation and decay.

Before an mRNA can be degraded, its poly(A) tail is removed by the Pan2-Pan3 and/or Ccr4-Not deadenylation complexes. This releases PABPC and may weaken the association of the eukaryotic translation initiation factor 4E (eIF4E) with the 5′-cap (purple circle). The decapping machinery can then access and remove the 5′-cap. Lsm1-7 can associate with oligo-A or 3′ uridylated tails to help recruit the decapping machinery. Decapping is followed by degradation of the mRNA in the 5′-3′ direction by Xrn1 or in the 3′-5′ direction by the cytoplasmic exosome.

Figure 4. Deadenylation by Pan2-Pan3 and Ccr4-Not.

A Sequential (or biphasic) model of deadenylation. In this model, Pan2-Pan3 preferentially removes the distal part of the poly(A) tail. A PABPC-interacting motif 2 (PAM2) within an intrinsically-disordered segment of Pan3 interacts with the C-terminal mademoiselle (MLLE) domain in PABPC. Ccr4-Not removes the poly(A) tail that is more proximal to the 3′-UTR. The most 5′ PABPC protein may be positioned on the poly(A) tail such that its RRM4 is located on the 3′-UTR of the mRNA. B Model of the Pan2-Pan3-Pab1-poly(A) complex. A cryoEM structure (PDB 6R5K) shows that Pan2-Pan3 contacts the interface between adjacent Pab1 molecules, providing an explanation for why it preferentially functions on longer poly(A) tails. In the structure, three Pab1 molecules are bound to a 90 nt poly(A) tail. C Ccr4 is a general deadenylase that degrades poly(A), even when it is bound by PABPC. Caf1 is a specialized deadenylase that degrades naked poly(A) and is blocked by PABPC.

Figure 5. Factors that influence deadenylation rate.

The deadenylation rate of Ccr4-Not is modulated by several factors. A RNA-binding proteins, such as Tristetraprolin (TTP) and Puf/Pumilio proteins act as RNA adapters to tether Ccr4-Not to specific transcripts, accelerating their deadenylation^,. TTP binds to AU-rich elements (AREs) in the 3′- untranslated region (UTR)^, and Puf proteins bind to Pumilio-recognition elements (PRE). B Slow translation elongation rate results in more rapid deadenylation, likely through direct interactions between Ccr4-Not and the ribosome. C RNA structure in the 3′- UTR and changes in the final nucleotides of the 3′-UTR influence deadenylation, possibly by altering Ccr4-Not or PABPC affinity for the 3′-UTR^,,,. D Insertion of nucleotides other than adenosine into the poly(A) tail can stall deadenylation by Ccr4-Not and Pan2- Pan3^,. In all panels, the 5′-cap is depicted as a purple circle; 5′-UTR is in yellow; coding sequence (CDS) is in orange; stop codon is a red hexagon; 3′-UTR is in red; and PABPC is in blue. Pan2-Pan3 deadenylation rate may also be influenced by the same factors.

Figure 6. Summary of recent insights into gene regulation by poly(A) tails.

Translation efficiency is depicted by ‘fast’ or ‘slow’ moving ribosomes on transcripts with optimal or non-optimal codons, respectively. Poly(A) tails can be short (~30 As) on stable, highly- translated mRNAs. The concentration of cytoplasmic poly(A)-binding protein (PABPC) may also affect the role of poly(A) tails in gene expression. Ccr4-Not may ′read’ translation elongation rate by detecting ribosomes containing empty A- and E-sites and this recruits the decapping machinery.