The long and the short of it: the role of the zinc finger polyadenosine RNA binding protein, Nab2, in control of poly(A) tail length

Abstract

In eukaryotic cells, addition of poly(A) tails to transcripts by 3'-end processing/polyadenylation machinery is a critical step in gene expression. The length of the poly(A) tail influences the stability, nuclear export and translation of mRNA transcripts. Control of poly(A) tail length is thus an important mechanism to regulate the abundance and ultimate translation of transcripts. Understanding the global regulation of poly(A) tail length will require dissecting the contributions of enzymes, regulatory factors, and poly(A) binding proteins (Pabs) that all cooperate to regulate polyadenylation. A recent addition to the Pab family is the CCCH-type zinc finger class of Pabs that includes S. cerevisiae Nab2 and its human counterpart, ZC3H14. In S. cerevisiae, Nab2 is an essential nuclear Pab implicated in both poly(A) RNA export from the nucleus and control of poly(A) tail length. Consistent with an important role in regulation of poly(A) tail length, depletion of Nab2 from yeast cells results in hyperadenylation of poly(A) RNA. In this review, we focus on the role of Nab2 in poly(A) tail length control and speculate on potential mechanisms by which Nab2 could regulate poly(A) tail length based on reported physical and genetic interactions. We present models, illustrating how Nab2 could regulate poly(A) tail length by limiting polyadenylation and/or enhancing trimming. Given that mutation of the gene encoding the human Nab2 homologue, ZC3H14, causes a form of autosomal recessive intellectual disability, we also speculate on how mutations in a gene encoding a ubiquitously expressed Pab lead specifically to neurological defects. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.

Fig. 1. Functional domains of Nab2

Nab2 is a 525 residue protein that possesses four key domains: an N-terminal domain (residues 1-97), a Q-rich domain (residues 104-169), an RGG domain (residues 201-261), and C-terminal tandem zinc finger domain (residues 262-473). The N-terminal domain of Nab2 (Nab2-N) facilitates nuclear export of poly(A) RNA and interacts with nuclear pore-associated Mlp1 protein in the nucleus and the nuclear rim-associated Gfd1 protein in the cytoplasm (20,22,28,30). The crystal structure of the N-terminal domain of Nab2 reveals that Nab2-N forms a five alpha-helix bundle with a proline-tryptophan-isoleucine (PWI)-like fold (20,22). The key residue Phe73 (red) that is important for interaction with Mlp1 and Tyr34 (magenta) that is critical for interaction with Gfd1 are highlighted (22,30,31). The RGG domain mediates interaction with the Nab2 import receptor, Kap104 (,–25). The Q-rich domain is not essential and currently has no characterized function (20). The C-terminal zinc finger domain contains seven tandem CCCH-type zinc fingers (ZnF) and mediates specific high affinity binding to polyadenosine RNA (10,20,21,26).

Fig. 2. Function of Nab2 in mRNA biogenesis

During RNA polymerase II (RNAPII) transcription, the THO transcription elongation complex (Tho2, Hpr1, Mft1, and Thp2), the ATPase, Sub2, the mRNA export cofactor, Yra1, the mRNA binding SR protein, Npl3, the poly(A) binding protein, Nab2, and other mRNA processing factors are all recruited to the nascent transcript (–8,38). Following capping and splicing, the polyadenylation signal (PAS) in the 3′-UTR of the transcript is recognized by the cleavage machinery, cleavage factor IA ((CFIA); Rna14, Rna15, Clp1 and Pcf11), cleavage factor IB ((CFIB); Hrp1/Nab4), and the cleavage and polyadenylation factor ((CPF); a complex including riboendonuclease, Ysh1/Brr5 (CPSF73 in mammals), the poly(A) polymerase, Pap1, and the Pap1 regulation factor, Fip1) (39). For cleavage at the poly(A) site (pA) in the 3′-UTR of the transcript, CFIA protein, Rna15, which contains a single RNA recognition motif (RRM), together with other CFIA subunits recognizes the A-rich positioning element (e.g. AAUAAN) in the PAS to position CPF to cleave the poly(A) site (–41). Hrp1, which contains two RRMs, binds to the AU-rich efficiency element (e.g. UAUAUAU) in the PAS and influences the efficiency of the cleavage reaction (39,42,43). Following cleavage, CPF-stimulated Pap1 processively synthesizes the poly(A) tail. During polyadenylation, Nab2 likely binds to the nascent poly(A) tail. Transcripts that are improperly processed (e.g. contain short poly(A) tails) due to defective 3′-end processing and polyadenylation are recognized, retained in the nucleus and degraded by the nuclear exosome riboexonuclease complex (Exo), containing the 3′-5′ riboexonuclease subunit, Rrp6 (3,5,51,52). Recognition of RNA targets by the exosome is facilitated by exosome cofactors, such as TRAMP (3,5,51,52). Together with the mRNA export cofactor, Yra1, Nab2 helps to recruit the heterodimeric mRNA export receptor, Mex67-Mtr2, to facilitate the nuclear export of the transcript (53). Nab2 also interacts with the nuclear pore complex-associated protein, Mlp1, to facilitate targeting of the transcript to the pore (22,28,30). Following Mex67-mediated export to the cytoplasm, Gfd1 helps tether Nab2 to the cytoplasmic face of the pore, while the RNA helicase, Dbp5, facilitates mRNP dissociation of Nab2, Npl3, Mex67, and other RNA-binding proteins from the transcript (29,31,37,54). During mRNP remodeling, the cytoplasmic Pab, Pab1, may bind to the poly(A) tail of the transcript to replace Nab2 in an exchange step. As Pab1 shuttles between the nucleus and cytoplasm and can bind the Rna15 CFIA cleavage factor, Pab1 likely initially loads onto the transcript in the nucleus (15,16,55,56). The cytoplasmic Pab1-bound transcript can then be translated or turned over. For cytoplasmic mRNA decay, the transcript is first deadenylated by the Ccr4-Not complex deadenylases, Ccr4 and Caf1. The transcript body is then decapped by Dcp2 and degraded from the 5′-end by the 5′-3′ riboexonuclease, Xrn1, or degraded from the 3′-end by the cytoplasmic exosome (4,5,58,59). Pab1 can also recruit the Pan2-Pan3 deadenylase complex (Pan2 is the deadenylase) to trim the poly(A) tails of specific transcripts (–63).

Fig. 3. Models for Nab2 control of poly(A) tail length

In a polyadenylation limiting model, Nab2 interacts with cleavage and polyadenylation factors (cleavage factor I (CFI); cleavage and polyadenylation factor (CPF)) or potentially alters the poly(A) RNA conformation to restrict poly(A) polymerase activity and limit poly(A) tail length. In the poly(A) tail trimming model, Nab2 recruits and modulates/stimulates a ribonuclease to trim the poly(A) tail to the correct length. Although, Nab2 is depicted with the cleavage and polyadenylation machinery during recruitment of a ribonuclease, Nab2 could also recruit a ribonuclease after polyadenylation at a later stage in mRNA biogenesis. These models are not necessarily mutually exclusive.

Fig. 4. Physical and genetic interactions of Nab2 with 3′-end processing and polyadenylation factors

Nab2 genetically interacts with cytoplasmic poly(A) binding protein, Pab1, poly(A) polymerase, Pap1, CFIA cleavage factor, Rna15, CPF subunit, Syc1, and nuclear exosome 3′-5′ riboexonuclease, Rrp6, and Ccr4-Not complex components, Not3, Caf40, and Caf130 (26,36,38,64). Nab2 physically interacts with the CFIB cleavage factor, Hrp1, and Ccr4-Not complex deadenylases, Ccr4 and Caf1, and component, Not5 (–67).