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Poly(A)-binding proteins (PABPs) regulate mRNA fate by controlling stability and translation through interactions with both the poly(A) tail and eIF4F complex. Many organisms have several paralogs of PABPs and eIF4F complex components and it is likely that different eIF4F/PABP complex combinations regulate distinct sets of mRNAs. Trypanosomes have five eIF4G paralogs, six of eIF4E and two PABPs, PABP1 and PABP2. Under starvation, polysomes dissociate and the majority of mRNAs, most translation initiation factors and PABP2 reversibly localise to starvation stress granules. To understand this more broadly we identified a protein interaction cohort for both T. brucei PABPs by cryo-mill/affinity purification-mass spectrometry. PABP1 very specifically interacts with the previously identified interactors eIF4E4 and eIF4G3 and few others. In contrast PABP2 is promiscuous, with a larger set of interactors including most translation initiation factors and most prominently eIF4G1, with its two partners TbG1-IP and TbG1-IP2. Only RBP23 was specific to PABP1, whilst 14 RNA-binding proteins were exclusively immunoprecipitated with PABP2. Significantly, PABP1 and associated proteins are largely excluded from starvation stress granules, but PABP2 and most interactors translocate to granules on starvation. We suggest that PABP1 regulates a small subpopulation of mainly small-sized mRNAs, as it interacts with a small and distinct set of proteins unable to enter the dominant pathway into starvation stress granules and localises preferentially to a subfraction of small polysomes. By contrast PABP2 likely regulates bulk mRNA translation, as it interacts with a wide range of proteins, enters stress granules and distributes over the full range of polysomes., Author summary Poly(A)-binding proteins (PABPs) bind to the poly(A) tails of eukaryotic mRNAs and function in regulating mRNA fate. Many eukaryotes have several PABP paralogs and the current view is that each PABP binds a specific subset of mRNAs. Trypanosoma brucei has two PABPs, and to understand the differential functionality of these paralogs we identified interacting proteins for each. We found unique interactors for both PABPs, and significant differences between the two interaction cohorts. Our data indicate that the two PABP paralogs of trypanosomes have very distinct roles in mediating mRNA fate.

Most Cryptococccus neoformans genes are interrupted by introns, and alternative splicing occurs very often. In this study, we examined the influence of introns on C. neoformans gene expression. For most tested genes, elimination of introns greatly reduces mRNA accumulation. Strikingly, the number and the position of introns modulate the gene expression level in a cumulative manner. A screen for mutant strains able to express functionally an intronless allele revealed that the nuclear poly(A) binding protein Pab2 modulates intron-dependent regulation of gene expression in C. neoformans. PAB2 deletion partially restored accumulation of intronless mRNA. In addition, our results demonstrated that the essential nucleases Rrp44p and Xrn2p are implicated in the degradation of mRNA transcribed from an intronless allele in C. neoformans. Double mutant constructions and over-expression experiments suggested that Pab2p and Xrn2p could act in the same pathway whereas Rrp44p appears to act independently. Finally, deletion of the RRP6 or the CID14 gene, encoding the nuclear exosome nuclease and the TRAMP complex associated poly(A) polymerase, respectively, has no effect on intronless allele expression., Author Summary Cryptococcus neoformans is a major human pathogen responsible for deadly infection in immunocompromised patients. The analysis of its genome previously revealed that most of its genes are interrupted by introns. Here, we demonstrate that introns modulate gene expression in a cumulative manner. We also demonstrate that introns can play a positive or a negative role in this process. We identify a nuclear poly(A) binding protein (Pab2p) as implicated in the intron-dependent control of gene expression in C. neoformans. We also demonstrate that the essential nucleases Rrp44p and Xrn2p are implicated in two independent pathways controlling the intron-dependent regulation of gene expression in C. neoformans. Xrn2p regulation seems to depend on Pab2p whereas Rrp44p acts independently. In contrast, the other exosome nuclease Rrp6p and the TRAMP associated poly(A) polymerase Cid14p do not appear to be implicated in this regulation. Our results provide new insights into the regulation of gene expression in eukaryotes and more specifically into the biology and virulence of C. neoformans.

The poly(A)-binding protein nuclear 1 (PABPN1) is a ubiquitously expressed protein that is thought to function during mRNA poly(A) tail synthesis in the nucleus. Despite the predicted role of PABPN1 in mRNA polyadenylation, little is known about the impact of PABPN1 deficiency on human gene expression. Specifically, it remains unclear whether PABPN1 is required for general mRNA expression or for the regulation of specific transcripts. Using RNA sequencing (RNA–seq), we show here that the large majority of protein-coding genes express normal levels of mRNA in PABPN1–deficient cells, arguing that PABPN1 may not be required for the bulk of mRNA expression. Unexpectedly, and contrary to the view that PABPN1 functions exclusively at protein-coding genes, we identified a class of PABPN1–sensitive long noncoding RNAs (lncRNAs), the majority of which accumulated in conditions of PABPN1 deficiency. Using the spliced transcript produced from a snoRNA host gene as a model lncRNA, we show that PABPN1 promotes lncRNA turnover via a polyadenylation-dependent mechanism. PABPN1–sensitive lncRNAs are targeted by the exosome and the RNA helicase MTR4/SKIV2L2; yet, the polyadenylation activity of TRF4-2, a putative human TRAMP subunit, appears to be dispensable for PABPN1–dependent regulation. In addition to identifying a novel function for PABPN1 in lncRNA turnover, our results provide new insights into the post-transcriptional regulation of human lncRNAs., Author Summary In eukaryotic cells, protein-coding genes are transcribed to produce pre-messenger RNAs (pre–mRNAs) that are processed at the 3′ end by the addition of a sequence of poly-adenosine. This 3′ end poly(A) tail normally confers positive roles to the mRNA life cycle by stimulating nuclear export and translation. The fundamental role of mRNA polyadenylation is generally mediated by the activity of poly(A)-binding proteins (PABPs) that bind to the 3′ poly(A) tail of eukaryotic mRNAs. In the nucleus, the evolutionarily conserved poly(A)-binding protein PABPN1 is thought to be important for gene expression, as it stimulates mRNA polyadenylation in biochemical assays. Using a high-throughput sequencing approach that quantitatively measures the level of RNA expressed from all genes, we addressed the global impact of a PABPN1 deficiency on human gene expression. Notably, we found that most mRNAs were normally expressed in PABPN1–deficient cells, a result inconsistent with a role for PABPN1 in general mRNA metabolism. Surprisingly, our genome-wide analysis unveiled a new function for PABPN1 in a polyadenylation-dependent pathway of RNA decay that targets non-protein coding genes. Our discovery that PABPN1 functions in the regulation of noncoding RNAs raises the possibility that oculopharyngeal muscular dystrophy, a disease associated with mutations in the PABPN1 gene, is caused by defective expression of noncoding RNAs.