Ribosomal Protein uS5 and Friends: Protein-Protein Interactions Involved in Ribosome Assembly and Beyond.

Ribosomal proteins are fundamental components of the ribosomes in all living cells. The ribosomal protein uS5 (Rps2) is a stable component of the small ribosomal subunit within all three domains of life. In addition to its interactions with proximal ribosomal proteins and rRNA inside the ribosome, uS5 has a surprisingly complex network of evolutionarily conserved non-ribosome-associated proteins. In this review, we focus on a set of four conserved uS5-associated proteins: the protein arginine methyltransferase 3 (PRMT3), the programmed cell death 2 (PDCD2) and its PDCD2-like (PDCD2L) paralog, and the zinc finger protein, ZNF277. We discuss recent work that presents PDCD2 and homologs as a dedicated uS5 chaperone and PDCD2L as a potential adaptor protein for the nuclear export of pre-40S subunits. Although the functional significance of the PRMT3-uS5 and ZNF277-uS5 interactions remain elusive, we reflect on the potential roles of uS5 arginine methylation by PRMT3 and on data indicating that ZNF277 and PRMT3 compete for uS5 binding. Together, these discussions highlight the complex and conserved regulatory network responsible for monitoring the availability and the folding of uS5 for the formation of 40S ribosomal subunits and/or the role of uS5 in potential extra-ribosomal functions.

PABPN1 prevents the nuclear export of an unspliced RNA with a constitutive transport element and controls human gene expression via intron retention.

Intron retention is a type of alternative splicing where one or more introns remain unspliced in a polyadenylated transcript. Although many viral systems are known to translate proteins from mRNAs with retained introns, restriction mechanisms generally prevent export and translation of incompletely spliced mRNAs. Here, we provide evidence that the human nuclear poly(A)-binding protein, PABPN1, functions in such restrictions. Using a reporter construct in which nuclear export of an incompletely spliced mRNA is enhanced by a viral constitutive transport element (CTE), we show that PABPN1 depletion results in a significant increase in export and translation from the unspliced CTE-containing transcript. Unexpectedly, we find that inactivation of poly(A)-tail exosome targeting by depletion of PAXT components had no effect on export and translation of the unspliced reporter mRNA, suggesting a mechanism largely independent of nuclear RNA decay. Interestingly, a PABPN1 mutant selectively defective in stimulating poly(A) polymerase elongation strongly enhanced the expression of the unspliced, but not of intronless, reporter transcripts. Analysis of RNA-seq data also revealed that PABPN1 controls the expression of many human genes via intron retention. Notably, PABPN1-dependent intron retention events mostly affected 3'-terminal introns and were insensitive to PAXT and NEXT deficiencies. Our findings thus disclose a role for PABPN1 in restricting nuclear export of intron-retained transcripts and reinforce the interdependence between terminal intron splicing, 3' end processing, and polyadenylation.

PDCD2 functions as an evolutionarily conserved chaperone dedicated for the 40S ribosomal protein uS5 (RPS2).

PDCD2 is an evolutionarily conserved protein with previously characterized homologs in Drosophila (zfrp8) and budding yeast (Tsr4). Although mammalian PDCD2 is essential for cell proliferation and embryonic development, the function of PDCD2 that underlies its fundamental cellular role has remained unclear. Here, we used quantitative proteomics approaches to define the protein-protein interaction network of human PDCD2. Our data revealed that PDCD2 specifically interacts with the 40S ribosomal protein uS5 (RPS2) and that the PDCD2-uS5 complex is assembled co-translationally. Loss of PDCD2 expression leads to defects in the synthesis of the small ribosomal subunit that phenocopy a uS5 deficiency. Notably, we show that PDCD2 is important for the accumulation of soluble uS5 protein as well as its incorporation into 40S ribosomal subunit. Our findings support that the essential molecular function of PDCD2 is to act as a dedicated ribosomal protein chaperone that recognizes uS5 co-translationally in the cytoplasm and accompanies uS5 to ribosome assembly sites in the nucleus. As most dedicated ribosomal protein chaperones have been identified in yeast, our study reveals that similar mechanisms exist in human cells to assist ribosomal proteins coordinate their folding, nuclear import and assembly in pre-ribosomal particles.

The 40S ribosomal protein uS5 (RPS2) assembles into an extraribosomal complex with human ZNF277 that competes with the PRMT3-uS5 interaction.

Ribosomal (r)-proteins are generally viewed as ubiquitous, constitutive proteins that simply function to maintain ribosome integrity. However, findings in the past decade have led to the idea that r-proteins have evolved specialized functions beyond the ribosome. For example, the 40S ribosomal protein uS5 (RPS2) is known to form an extraribosomal complex with the protein arginine methyltransferase PRMT3 that is conserved from fission yeast to humans. However, the full scope of uS5's extraribosomal functions, including whether uS5 interacts with any other proteins, is not known. In this study, we identify the conserved zinc finger protein 277 (ZNF277) as a new uS5-associated protein by using quantitative proteomics approaches in human cells. As previously shown for PRMT3, we found that ZNF277 uses a C2H2-type zinc finger domain to recognize uS5. Analysis of protein-protein interactions in living cells indicated that the ZNF277-uS5 complex is found in the cytoplasm and the nucleolus. Furthermore, we show that ZNF277 and PRMT3 compete for uS5 binding, because overexpression of PRMT3 inhibited the formation of the ZNF277-uS5 complex, whereas depletion of cellular ZNF277 resulted in increased levels of uS5-PRMT3. Notably, our results reveal that ZNF277 recognizes nascent uS5 in the course of mRNA translation, suggesting cotranslational assembly of the ZNF277-uS5 complex. Our findings thus unveil an intricate network of evolutionarily conserved protein-protein interactions involving extraribosomal uS5, suggesting a key role for uS5 beyond the ribosome.

Human PDCD2L Is an Export Substrate of CRM1 That Associates with 40S Ribosomal Subunit Precursors.

Protein arginine methyltransferase 3 (PRMT3) forms a stable complex with 40S ribosomal protein S2 (RPS2) and contributes to ribosome biogenesis. However, the molecular mechanism by which PRMT3 influences ribosome biogenesis and/or function still remains unclear. Using quantitative proteomics, we identified human programmed cell death 2-like (PDCD2L) as a novel PRMT3-associated protein. Our data suggest that RPS2 promotes the formation of a conserved extraribosomal complex with PRMT3 and PDCD2L. We also show that PDCD2L associates with 40S subunit precursors that contain a 3'-extended form of the 18S rRNA (18S-E pre-rRNA) and several pre-40S maturation factors. PDCD2L shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner using a leucine-rich nuclear export signal that is sufficient to direct the export of a reporter protein. Although PDCD2L is not required for the biogenesis and export of 40S ribosomal subunits, we found that PDCD2L-null cells accumulate free 60S ribosomal subunits, which is indicative of a deficiency in 40S subunit availability. Our data also indicate that PDCD2L and its paralog, PDCD2, function redundantly in 40S ribosomal subunit production. Our findings uncover the existence of an extraribosomal complex consisting of PDCD2L, RPS2, and PRMT3 and support a role for PDCD2L in the late maturation of 40S ribosomal subunits.

Polyadenylation-dependent control of long noncoding RNA expression by the poly(A)-binding protein nuclear 1.

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.