A nuclear 3'-5' exonuclease involved in mRNA degradation interacts with Poly(A) polymerase and the hnRNA protein Npl3p

Inactivation of poly(A) polymerase (encoded by PAP1) in Saccharomyces cerevisiae cells carrying the temperature-sensitive, lethal pap1-1 mutation results in reduced levels of poly(A)(+) mRNAs. Genetic selection for suppressors of pap1-1 yielded two recessive, cold-sensitive alleles of the gene RRP6. These suppressors, rrp6-1 and rrp6-2, as well as a deletion of RRP6, allow growth of pap1-1 strains at high temperature and partially restore the levels of poly(A)(+) mRNA in a manner distinct from the cytoplasmic mRNA turnover pathway and without slowing a rate-limiting step in mRNA decay. Subcellular localization of an Rrp6p-green fluorescent protein fusion shows that the enzyme residues in the nucleus. Phylogenetic analysis and the nature of the rrp6-1 mutation suggest the existence of a highly conserved 3'-5' exonuclease core domain within Rrp6p. As predicted, recombinant Rrp6p catalyzes the hydrolysis of a synthetic radiolabeled RNA in a manner consistent with a 3'-5' exonucleolytic mechanism. Genetic and biochemical experiments indicate that Rrp6p interacts with poly(A) polymerase and with Npl3p, a poly(A)(+) mRNA binding protein implicated in pre-mRNA processing and mRNA nuclear export. These findings suggest that Rrp6p may interact with the mRNA polyadenylation system and thereby play a role in a nuclear pathway for the degradation of aberrantly processed precursor mRNAs.     

The RNA binding domains of the nuclear poly(A)-binding protein

The nuclear poly(A)-binding protein (PABPN1) is involved in the synthesis of the mRNA poly(A) tails in most eukaryotes. We report that the protein contains two RNA binding domains, a ribonucleoprotein-type RNA binding domain (RNP domain) located approximately in the middle of the protein sequence and an arginine-rich C-terminal domain. The C-terminal domain also promotes self-association of PABPN1 and moderately cooperative binding to RNA. Whereas the isolated RNP domain binds specifically to poly(A), the isolated C-terminal domain binds non-specifically to RNA and other polyanions. Despite this nonspecific RNA binding by the C-terminal domain, selection experiments show that adenosine residues throughout the entire minimal binding site of approximately 11 nucleotides are recognized specifically. UV-induced cross-links with oligo(A) carrying photoactivatable nucleotides at different positions all map to the RNP domain, suggesting that most or all of the base-specific contacts are made by the RNP domain, whereas the C-terminal domain may contribute nonspecific contacts, conceivably to the same nucleotides. Asymmetric dimethylation of 13 arginine residues in the C-terminal domain has no detectable influence on the interaction of the protein with RNA. The N-terminal domain of PABPN1 is not required for RNA binding but is essential for the stimulation of poly(A) polymerase.     

Control of poly(A) polymerase level is essential to cytoplasmic polyadenylation and early development in Drosophila

Poly(A) polymerase (PAP) has a role in two processes, polyadenylation of mRNA precursors in the nucleus and translational control of certain mRNAs by cytoplasmic elongation of their poly(A) tails, particularly during early development. It was found recently that at least three different PAP genes exist in mammals, encoding several PAP isoforms. The in vivo specificity of function of each PAP isoform currently is unknown. Here, we analyse PAP function in Drosophila: We show that a single PAP isoform exists in Drosophila that is encoded by the hiiragi gene. This single Drosophila PAP is active in specific polyadenylation in vitro and is involved in both nuclear and cytoplasmic polyadenylation in vivo. Therefore, the same PAP can be responsible for both processes. In addition, in vivo overexpression of PAP does not affect poly(A) tail length during nuclear polyadenylation, but leads to a dramatic elongation of poly(A) tails and a loss of specificity during cytoplasmic polyadenylation, resulting in embryonic lethality. This demonstrates that regulation of the PAP level is essential for controlled cytoplasmic polyadenylation and early development.     

Accelerated poly(A) loss on alpha-tubulin mRNAs during protein synthesis inhibition in Chlamydomonas

Detachment of flagella in Chlamydomonas reinhardii stimulates a rapid accumulation of tubulin mRNAs. The induced tubulin mRNAs are normally rapidly degraded following flagellar regeneration, but inhibition of protein synthesis with cycloheximide prevents their degradation. alpha-Tubulin poly(A) tail lengths were measured during normal accumulation and degradation, and in cycloheximide-treated cells. To measure alpha-tubulin mRNA poly(A) chain lengths with high resolution, specific 3' fragments of alpha 1- and alpha 2-tubulin mRNAs, generated by RNase H digestion of mRNA-oligonucleotide hybrids, were sized by Northern analysis. Both alpha-tubulin mRNAs have a newly synthesized poly(A) chain of about 110 adenylate residues. The poly(A) tails shorten with time, and show an average length of 40 to 60 adenylate residues by 90 minutes after deflagellation, at which time induced alpha-tubulin mRNA is being rapidly degraded. Poly(A) loss is significantly accelerated in cycloheximide-treated cells, and this loss is not attributible simply to the longer time the stabilized molecules spend in the cytoplasm. A large fraction of alpha-tubulin mRNA accumulates as mRNA with very short poly(A) tails (less than 10 residues) in the presence of cycloheximide, indicating that deadenylated alpha-tubulin mRNAs can be stable in vivo, at least in the absence of protein synthesis. The rate and extent of poly(A) loss in cycloheximide are greater for alpha 2-tubulin mRNA than for alpha 1-tubulin mRNA. This difference cannot be attributed to differential ribosome loading. This finding is interesting in that the two mRNAs are very similar in sequence with the exception of their 3' untranslated regions.     

Functional significance of the interaction between the mRNA-binding protein, Nab2, and the nuclear pore-associated protein, Mlp1, in mRNA export

Nuclear export of mRNA requires several key mRNA-binding proteins that recognize and remodel the mRNA and target it for export via interactions with the nuclear pore complex. In Saccharomyces cerevisiae, the shuttling heterogeneous nuclear ribonucleoprotein, Nab2, which is essential for mRNA export, specifically recognizes poly(A) RNA and binds to the nuclear pore-associated protein, myosin-like protein 1 (Mlp1), which functions in mRNA export and quality control. Specifically, the N-terminal domain of Nab2 (Nab2-N; residues 1-97) interacts directly with the C-terminal globular domain of Mlp1 (CT-Mlp1: residues 1490-1875). Recent structural and binding studies focused on Nab2-N have shown that Nab2-N contains a hydrophobic patch centered on Phe(73) that is critical for interaction with Mlp1. Engineered amino acid changes within this patch disrupt the Nab2/Mlp1 interaction in vitro. Given the importance of Nab2 and Mlp1 to mRNA export, we have examined the Nab2/Mlp1 interaction in greater detail and analyzed the functional consequences of disrupting the interaction in vivo. We find that the Nab2-binding domain of Mlp1 (Mlp1-NBD) maps to a 183-residue region (residues 1586-1768) within CT-Mlp1, binds directly to Nab2 with micromolar affinity, and confers nuclear accumulation of poly(A) RNA. Furthermore, we show that cells expressing a Nab2 F73D mutant that cannot interact with Mlp1 exhibit nuclear accumulation of poly(A) RNA and that this nab2 F73D mutant genetically interacts with alleles of two essential mRNA export genes, MEX67 and YRA1. These data provide in vivo evidence for a model of mRNA export in which Nab2 is important for targeting mRNAs to the nuclear pore for export.     

Two separate regions essential for nuclear import of the hnRNP D nucleocytoplasmic shuttling sequence

Heterogeneous nuclear ribonucleoprotein (hnRNP) D/AUF1 functions in mRNA genesis in the nucleus and modulates mRNA decay in the cytoplasm. Although it is primarily nuclear, it shuttles between the nucleus and cytoplasm. We studied the nuclear import and export of the last exon-encoding sequence common to all its isoforms by its expression as a green fluorescent protein-fusion protein in HeLa cells and by heterokaryon assay. The C-terminal 19-residue sequence (SGYGKVSRRGGHQNSYKPY) was identified as an hnRNP D nucleocytoplasmic shuttling sequence (DNS). In vitro nuclear transport using permeabilized cells indicated that nuclear import of DNS is mediated by transportin-1 (Trn-1). DNS accumulation in the nucleus was dependent on Trn-1, Ran, and energy in multiple rounds of nuclear transport. Use of DNS with deletions, alanine scanning mutagenesis and point mutations revealed that two separate regions (the N-terminal seven residues and the C-terminal two residues) are crucial for in vivo and in vitro transport as well as for interaction with Trn-1. The N- and C-terminal motifs are conserved in the shuttling sequences of hnRNP A1 and JKTBP.     

Interaction of poly(A) polymerase with the 25-kDa subunit of cleavage factor I.

Mammalian poly(A) polymerase (PAP), a key enzyme in the pre-mRNA 3'-end processing reaction, carries the catalytic domain in the N-terminal region, an RNA binding domain, two nuclear localization signals, and a serine/threonine-rich regulatory domain in the C-terminal region. Using LexA-based yeast two-hybrid screening, we identified a cDNA encoding the 25-kDa subunit of cleavage factor I (CFI-25) as a protein that interacts with the C-terminal region of mouse PAP. The glutathione S-transferase pull-down assay and the immunoprecipitation experiment revealed that PAP directly interacts with CFI-25 and that the C-terminal 69 residues of PAP and the N-terminal 60 residues of CFI-25 are sufficient for the interaction between CFI-25 and PAP. Since CFI is known to function in the assembly of the pre-mRNA 3'-processing complex, this interaction may play an important role in the assembly of the processing complex and/or in the regulation of PAP activity within the complex.     

RNA turnover: unexpected consequences of being tailed

In eukaryotic cells, the 3' poly(A) tails found on mRNA influence their stability and translation. The discovery of a second nuclear poly(A) polymerase complex has fueled a series of reports defining a new and unexpected role for 3' end poly(A) tails in the nuclear surveillance and turnover of noncoding RNAs and intergenic mRNAs of unknown function.     

Influenza A virus NS1 protein targets poly(A)-binding protein II of the cellular 3'-end processing machinery

Influenza A virus NS1 protein (NS1A protein) via its effector domain targets the poly(A)-binding protein II (PABII) of the cellular 3'-end processing machinery. In vitro the NS1A protein binds the PABII protein, and in vivo causes PABII protein molecules to relocalize from nuclear speckles to a uniform distribution throughout the nucleoplasm. In vitro the NS1A protein inhibits the ability of PABII to stimulate the processive synthesis of long poly(A) tails catalyzed by poly(A) polymerase (PAP). Such inhibition also occurs in vivo in influenza virus-infected cells, where the NS1A protein via its effector domain causes the nuclear accumulation of cellular pre-mRNAs which contain short ( approximately 12 nucleotide) poly(A) tails. Consequently, although the NS1A protein also binds the 30 kDa subunit of the cleavage and polyadenylation specificity factor (CPSF), 3' cleavage of some cellular pre-mRNAs still occurs in virus-infected cells, followed by the PAP-catalyzed addition of short poly(A) tails. Subsequent elongation of these short poly(A) tails is blocked because the NS1A protein inhibits PABII function. Nuclear-cytoplasmic shuttling of PABII, an activity implicating this protein in the nuclear export of cellular mRNAs, is also inhibited by the NS1A protein. In vitro assays suggest that the 30 kDa CPSF and PABII proteins bind to non-overlapping regions of the NS1A protein effector domain and indicate that these two 3' processing proteins also directly bind to each other.     

An essential cytoplasmic function for the nuclear poly(A) binding protein, PABP2, in poly(A) tail length control and early development in Drosophila

Translational control of maternal mRNA through regulation of poly(A) tail length is crucial during early development. The nuclear poly(A) binding protein, PABP2, was identified biochemically from its role in nuclear polyadenylation. Here, we analyze the in vivo function of PABP2 in Drosophila. PABP2 is required in vivo for polyadenylation, and Pabp2 function, including poly(A) polymerase stimulation, is essential for viability. We also demonstrate an unanticipated cytoplasmic function for PABP2 during early development. In contrast to its role in nuclear polyadenylation, cytoplasmic PABP2 acts to shorten the poly(A) tails of specific mRNAs. PABP2, together with the deadenylase CCR4, regulates the poly(A) tails of oskar and cyclin B mRNAs, both of which are also controlled by cytoplasmic polyadenylation. Both Cyclin B protein levels and embryonic development depend upon this regulation. These results identify a regulator of maternal mRNA poly(A) tail length and highlight the importance of this mode of translational control.     

Two motifs essential for nuclear import of the hnRNP A1 nucleocytoplasmic shuttling sequence M9 core

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 regulates mRNA genesis. It shuttles between the nucleus and cytoplasm. Its shuttling signal is a 38-residue sequence M9. We studied the nuclear import and export of M9 by mutational analysis. Heterokaryon assay indicated that the 19-residue sequence SNFGPMKGGNFGGRSSGPY (M9 core) is necessary and sufficient for shuttling. Moreover, M9 core mutation revealed that in addition to the hitherto characterized N-terminal motif SNFGPMK, the C-terminal motif PY is crucial for nuclear import as well as for binding to transportin. Key residues of the motifs are conserved in the shuttling signals of hnRNP D and JKTBP.     

ERK is a novel regulatory kinase for poly(A) polymerase

Poly(A) polymerase (PAP), which adds poly(A) tails to the 3′ end of mRNA, can be phosphorylated at several sites in the C-terminal domain. Phosphorylation often mediates regulation by extracellular stimuli, suggesting PAP may be regulated by such stimuli. In this study, we found that phosphorylation of PAP was increased upon growth stimulation and that the mitogen-activated protein kinase ERK was responsible for the increase in phosphorylation. We identified serine 537 of PAP as a unique phosphorylation site by ERK. PAP phosphorylation of serine 537 by ERK increased its nonspecific polyadenylation activity in vitro. This PAP activity was also activated by stimulation of ERK with phorbol-12-myristate-13-acetate in vivo. These data suggest that ERK is a novel regulatory kinase for PAP and further, that PAP activity could be regulated by extracellular stimuli through an ERK-dependent signaling pathway(s).