R3H Domain Research Articles

Comprehensive pan-cancer analysis and experiments revealed R3HDM1 as a novel predictive biomarker for prognosis and immune therapy response.

R3HDM1, an RNA binding protein with one R3H domain, remains uncharacterized in terms of its association with tumor progression, malignant cell regulation, and the tumor immune microenvironment. This paper aims to fill this gap by analyzing the potential of R3HDM1 in diagnosis, prognosis, chemotherapy, and immune function across various cancers. Data was collected from the Firehost database (http://gdac.broadinstitute.org) to obtain the TCGA pan-cancer queue containing tumor and normal samples. Additional data on miRNA, TCPA, mutations, and clinical information were gathered from the UCSC Xena database (https://xenabrowser.net/datapages/). The mutation frequency and locus of R3HDM1 in the TCGA database were examined using the cBioPortal. External validation through GEO data was conducted to assess the differential expression of R3HDM1 in different cancers. Protein expression levels were evaluated using the Clinical Proteomics Tumor Analysis Alliance (CPTAC). The differential expression of R3HDM1 was verified in lung adenocarcinoma cell lines and normal lung glandular epithelial cells via RT-qPCR. Cell migration and proliferation experiments were conducted by knocking down the expression of R3HDM1 in two lung adenocarcinoma cell lines using small interfering RNA. The biological role of R3HDM1 in pan-cancer was explored using the GSEA method. Multiple immune infiltration algorithms from the TIMER2.0 database was employed to investigate the correlation between R3HDM1 expression and the tumor immune microenvironment. Validation of transcriptome immune infiltration was based on 140 single-cell datasets from the TISCH database. The study also characterized a pan-cancer survival profile and analyzed the differential expression of R3HDM1 in different molecular subtypes. The relationship between R3HDM1 and drug resistance was investigated using four chemotherapy data sources: CellMiner, GDSC, CTRP and PRISM. The impact of chemicals on the expression of R3HDM1 was explored through the CTD database. The study revealed differential expression of R3HDM1 in various tumors, indicating its potential as an early diagnostic marker. Changes in somatic copy number (SCNA) and DNA methylation were identified as factors contributing to abnormal expression levels. Additionally, the study found that R3HDM1 expression is associated with clinical features, metabolic pathways, and important pathways related to metastasis and the immune system. High expression of R3HDM1 was linked to poor prognosis across different tumors and altered drug sensitivity. Furthermore, the expression of R3HDM1 showed significant correlations with immune modulatory molecules and biomarkers of lymphocyte subpopulation infiltration. Finally, the study highlighted four chemicals that could influence the expression of R3HDM1. Overall, this study proposes that R3HDM1 expression is a promising biomarker for predicting the prognosis of cancer, especially lung adenocarcinoma, and the efficacy of immunotherapy, demonstrating the rationale for further exploration in the development of anti-tumor therapies.

The thymocyte-specific RNA-binding protein Arpp21 provides TCR repertoire diversity by binding to the 3’-UTR and promoting Rag1 mRNA expression

The regulation of thymocyte development by RNA-binding proteins (RBPs) is largely unexplored. We identify 642 RBPs in the thymus and focus on Arpp21, which shows selective and dynamic expression in early thymocytes. Arpp21 is downregulated in response to T cell receptor (TCR) and Ca2+ signals. Downregulation requires Stim1/Stim2 and CaMK4 expression and involves Arpp21 protein phosphorylation, polyubiquitination and proteasomal degradation. Arpp21 directly binds RNA through its R3H domain, with a preference for uridine-rich motifs, promoting the expression of target mRNAs. Analysis of the Arpp21–bound transcriptome reveals strong interactions with the Rag1 3′-UTR. Arpp21–deficient thymocytes show reduced Rag1 expression, delayed TCR rearrangement and a less diverse TCR repertoire. This phenotype is recapitulated in Rag1 3′-UTR mutant mice harboring a deletion of the Arpp21 response region. These findings show how thymocyte-specific Arpp21 promotes Rag1 expression to enable TCR repertoire diversity until signals from the TCR terminate Arpp21 and Rag1 activities.

Association between a single nucleotide polymorphism in the R3HCC1 gene and irinotecan toxicity.

Irinotecan is a useful anticancer drug for colorectal cancer treatment. UGT1A1*28 and *6 gene polymorphisms are known risk factors for irinotecan-associated toxicity. However, severe adverse effects due to irinotecan have been observed even in patients who do not harbor UGT1A1*28 or *6. We investigated gene polymorphisms in the whole exome to identify useful biomarkers for irinotecan toxicity other than UGT1A. A total of 178 patients with metastatic colorectal cancer (mCRC) and 87 patients with pancreatic cancer were treated with FOLFIRI, FOLFOX, FOLFOXIRI, modified FOLFIRINOX, or gemcitabine plus nab-paclitaxel. Genome-wide screening was performed using whole-exome sequencing (WES), and validation analysis was performed using qPCR with a hydrolysis probe. Using WES after a doublet chemotherapy regimen comprising irinotecan and 5-fluorouracil (n=15), seven single nucleotide polymorphisms (SNPs) were identified as candidate biomarkers for irinotecan-associated toxicity of neutropenia. Among the seven SNPs, an SNP in R3H domain and coiled-coil containing 1 (R3HCC1; c.919G > A, rs2272761) showed a significant association with neutropenia (>grade 3) after doublet chemotherapy. Patients receiving irinotecan including triplet chemotherapy, FOLFOXIRI for mCRC (n=23) or modified FOLFIRINOX for pancreatic cancer (n=40), also showed significant linear trends between R3HCC1 polymorphism and neutropenia (p=0.017 and 0.046, respectively). No significant association was observed in patients treated with irinotecan-free regimens, FOLFOX for mCRC (n=66), and gemcitabine plus nab-paclitaxel for pancreatic cancer (n=47). Thus, an SNP in the R3HCC1 gene may be a useful biomarker for the toxicity of irinotecan-containing chemotherapy for mCRC and pancreatic cancer.

Assembly of RNA polymerase III complex involves a putative co-translational mechanism

Detailed knowledge of structures of yeast RNA polymerases (RNAPs) contrasts with the limited information that is available on the control of their assembly. RNAP enzymes are large heteromeric complexes that function in the nucleus, but they are assembled in the cytoplasm and imported to the nucleus with help from specific auxiliary factors. Here, I review a recent study that suggests that the formation of an early-stage assembly intermediate of the RNAP III complex occurs through a co-translational mechanism. According to our hypothesis, RNAP III assembly might be seeded while the Rpb10 subunit of the enzyme core is being synthesized by cytoplasmic ribosome machinery. The co-translational assembly of RNAP III is mediated by Rbs1 protein which binds to 3′-untranslated regions in mRNA in a way that depends on the R3H domain in the Rbs1 sequence.

Rbs1 protein, involved in RNA polymerase III complex assembly in the yeast Saccharomyces cerevisiae, induces a Gcn4 response and forms aggregates when overproduced.

We previously reported the function of Rbs1 protein in RNA polymerase III complex assembly via interactions with both, proteins and mRNAs. Rbs1 is a poly(A)-binding protein. The R3H domain in Rbs1 is required for mRNA interactions. The present study utilized the results of a genome-wide analysis of RNA binding by Rbs1 to show a direct interaction between Rbs1 with the 5'-untranslated region (5'-UTR) in PCL5 mRNA. By examining Pcl5 protein levels, we found that Rbs1 overproduction inhibited the translation of PCL5 mRNA. Pcl5 is a cyclin that is associated with Pho85 kinase, which is involved in the degradation of Gcn4 transcription factor. Consequently, lower levels of Pcl5 that resulted from Rbs1 overproduction increased the Gcn4 response. The functional R3H domain in Rbs1 was required for the downregulation of Pcl5 translation and increase in the Gcn4 response, thus validating a regulatory mechanism that relies on the interaction between Rbs1 and the 5'-UTR in PCL5 mRNA. Rbs1 protein was further characterized by microscopy, which identified single Rbs1 assemblies in part of the cell population. The presence of Rbs1 aggregates was confirmed by the fractionation of cellular extracts. Altogether, our results suggest a more general role of Rbs1 in regulating cellular metabolism beyond the assembly of RNA polymerase III.

R3HDM1 haploinsufficiency is associated with mild intellectual disability.

R3HDM1 (R3H domain containing 1) is an uncharacterized RNA-binding protein that is highly expressed in the human cerebral cortex. We report the first case of a 12-year-old Japanese male with haploinsufficiency of R3HDM1. He presented with mild intellectual disability (ID) and developmental delay. He had a pericentric inversion of 46,XY,inv(2)(p16.1q21.3)dn with breakpoints in intron 19 of R3HDM1 (2q21.3) and the intergenic region (2p16.1). The R3HDM1 levels in his lymphoblastoid cells were reduced to approximately half that of the healthy controls. However, the expression of MIR128-1, in intron 18 of R3HDM1, was not affected via the pericentric inversion. Knockdown of R3HDM1 in mouse embryonic hippocampal neurons suppressed dendritic growth and branching. Notably, the Database of Genomic Variants reported the case of a healthy control with a 488-kb deletion that included both R3HDM1 and MIR128-1. miR-128 has been reported to inhibit dendritic growth and branching in mouse brain neurons, which directly opposes the novel functions of R3HDM1. These findings suggest that deleting both R3HDM1 and MIR128-1 alleviates the symptoms of the disease caused by loss-of-function mutations in R3HDM1 only. Thus, haploinsufficiency of R3HDM1 in the patient may be the cause of the mild ID due to the genetic imbalance between R3HDM1 and MIR128-1.

A novel podocyte protein, R3h domain containing-like, inhibits TGF-β-induced p38 MAPK and regulates the structure of podocytes and glomerular basement membrane.

Not only in kidney glomerular physiological function but also glomerular pathology especially in diabetic condition, glomerular podocytes play pivotal roles. Therefore, it is important to increase our knowledge about the genes and proteins expressed in podocytes. Recently, we have identified a novel podocyte-expressed gene, R3h domain containing-like (R3hdml) and analyzed its function in vivo as well as in vitro. Transforming growth factor-β (TGF-β) signaling regulated the expression of R3hdml. And R3hdml inhibited p38 mitogen-activated protein kinase phosphorylation, which was induced by TGF-β, leading to the amelioration of podocyte apoptosis. Furthermore, a lack of R3hdml in mice significantly worsened glomerular function in streptozotocin (STZ)-induced diabetes, while overexpression of R3hdml ameliorated albuminuria in STZ-induced diabetes. Our results surmise that the functional analyses of R3hdml may lead to the development of novel therapeutic strategies for diabetic nephropathy in the future. KEY MESSAGES: • A novel podocyte expressed protein R3h domain containing-like was identified. • R3HDML inhibits podocyte apoptosis by inhibiting TGF-β-mediated p38 MAPK signaling. • Overexpression of R3HDML ameliorates albuminuria in STZ-induced diabetes mice. • R3HDML may prove to be a novel therapeutic strategy for diabetic nephropathy.

The expression of Rpb10, a small subunit common to RNA polymerases, is modulated by the R3H domain-containing Rbs1 protein and the Upf1 helicase.

The biogenesis of eukaryotic RNA polymerases is poorly understood. The present study used a combination of genetic and molecular approaches to explore the assembly of RNA polymerase III (Pol III) in yeast. We identified a regulatory link between Rbs1, a Pol III assembly factor, and Rpb10, a small subunit that is common to three RNA polymerases. Overexpression of Rbs1 increased the abundance of both RPB10 mRNA and the Rpb10 protein, which correlated with suppression of Pol III assembly defects. Rbs1 is a poly(A)mRNA-binding protein and mutational analysis identified R3H domain to be required for mRNA interactions and genetic enhancement of Pol III biogenesis. Rbs1 also binds to Upf1 protein, a key component in nonsense-mediated mRNA decay (NMD) and levels of RPB10 mRNA were increased in a upf1Δ strain. Genome-wide RNA binding by Rbs1 was characterized by UV cross-linking based approach. We demonstrated that Rbs1 directly binds to the 3′ untranslated regions (3′UTRs) of many mRNAs including transcripts encoding Pol III subunits, Rpb10 and Rpc19. We propose that Rbs1 functions by opposing mRNA degradation, at least in part mediated by NMD pathway. Orthologues of Rbs1 protein are present in other eukaryotes, including humans, suggesting that this is a conserved regulatory mechanism.

R3hdml regulates satellite cell proliferation and differentiation.

In this study, we identified a previously uncharacterized skeletal satellite cell-secreted protein, R3h domain containing-like (R3hdml). Expression of R3hdml increases during skeletal muscle development and differentiation in mice. Body weight and skeletal muscle mass of R3hdml knockout (KO) mice are lower compared to control mice. Expression levels of cell cycle-related markers, phosphorylation of Akt, and expression of insulin-like growth factor within the skeletal muscle are reduced in R3hdml KO mice compared to control mice. Expression of R3hdml increases during muscle regeneration in response to cardiotoxin (CTX)-induced muscle injury. Recovery of handgrip strength after CTX injection was significantly impaired in R3hdml KO mice, which is rescued by R3hdml. Our results indicate that R3hdml is required for skeletal muscle development, regeneration, and, in particular, satellite cell proliferation and differentiation.

Screening and identification of biomarkers associated with clinicopathological parameters and prognosis in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a major type of malignant tumor of the oral cavity. Despite marked advances in the management and diagnosis of OSCC, the associated overall survival ratio has only exhibited a modest increase in recent years. The present study aimed to identify potential crucial genes associated with clinical features and prognosis for OSCC, and to provide a basis for further investigation. RNA-sequencing data and corresponding clinical information were downloaded from The Cancer Genome Atlas database and differentially expressed mRNAs (DEmRNAs) were identified using the edgeR package. Bioinformatics analysis was performed to identify differentially expressed clinical features-associated mRNAs (CFmRNAs) and enhance the current knowledge of the function of them. Functional enrichment analysis and protein-protein interplay (PPI) network analysis were then performed to better understand CFmRNAs. Survival-associated genes were analyzed with Kaplan-Meier survival curves and the log-rank test. A total of 2,013 DEmRNAs between OSCC samples and normal tissues were identified, 180 of which were associated with clinical features. A total of 17 GO terms and 4 KEGG pathways were significantly enriched in functional enrichment analysis. A total of 4 hub genes (albumin, statherin, neurotensin and mucin 7) were identified in the PPI network analysis. A total of 6 genes (DDB1 and CUL4 associated factor 4 like 2, opiorphin prepropeptide, R3H domain containing like, transmembrane phosphatase with tensin homology, actin like 8 and protocadherin α 11) were observed to have an influence on survival. The DEmRNAs identified may have a crucial role in the genesis and development of OSCC and may be further developed for diagnostic, therapeutic and prognostic applications for OSCC in the future.

Contributions of the C-terminal domain to poly(A)-specific ribonuclease (PARN) stability and self-association

Poly(A)-specific ribonuclease (PARN) catalyzes the degradation of mRNA poly(A) tail to regulate translation efficiency and mRNA decay in higher eukaryotic cells. The full-length PARN is a multi-domain protein containing the catalytic nuclease domain, the R3H domain, the RRM domain and the C-terminal intrinsically unstructured domain (CTD). The roles of the three well-structured RNA-binding domains have been extensively studied, while little is known about CTD. In this research, the impact of CTD on PARN stability and aggregatory potency was studied by comparing the thermal inactivation and denaturation behaviors of full-length PARN with two N-terminal fragments lacking CTD. Our results showed that K+ induced additional regular secondary structures and enhanced PARN stability against heat-induced inactivation, unfolding and aggregation. CTD prevented PARN from thermal inactivation but promoted thermal aggregation to initiate at a temperature much lower than that required for inactivation and unfolding. Blue-shift of Trp fluorescence during thermal transitions suggested that heat treatment induced rearrangements of domain organizations. CTD amplified the stabilizing effect of K+, implying the roles of CTD was mainly achieved by electrostatic interactions. These results suggested that CTD might dynamically interact with the main body of the molecule and release of CTD promoted self-association via electrostatic interactions.

Self-association of poly(A)-specific ribonuclease (PARN) triggered by the R3H domain

Poly(A)-specific ribonuclease (PARN) is a deadenylase with three RNA-binding domains (the nuclease, R3H and RRM domains) and a C-terminal domain. PARN participates in diverse physiological processes by regulating mRNA fates through deadenylation. PARN mainly exists as a dimer in dilute solutions. In this research, we found that PARN could self-associate into tetramer and high-order oligomers both in vitro and in living cells. Mutational and spectroscopic analysis indicated that PARN oligomerization was triggered by the R3H domain, which led to the solvent-exposed Trp219 fluorophore to become buried in a solvent-inaccessible microenvironment. The RRM and C-terminal domains also played a role in modulating the dissociation rate of the tetrameric PARN. Enzymatic analysis indicated that tetramerization did not affect the catalytic behavior of the full-length PARN and truncated enzymes containing the RRM domain, which might be caused by the high propensity of the dimeric proteins to self-associate into oligomers. Tetramerization significantly enhanced the catalytic activity and processivity of the truncated form with the removal of the RRM and C-terminal domains. The results herein suggested that self-association might be one of the regulation methods for PARN to achieve a highly regulated deadenylase activity. We propose that self-association may facilitate PARN to concentrate around the target mRNAs by restricted diffusion.

Distinct roles of the R3H and RRM domains in poly(A)-specific ribonuclease structural integrity and catalysis

Deadenylases specifically catalyze the degradation of eukaryotic mRNA poly(A) tail in the 3′- to 5′-end direction with the release of 5′-AMP as the product. Among the deadenylase family, poly(A)-specific ribonuclease (PARN) is unique in its domain composition, which contains three potential RNA-binding domains: the catalytic nuclease domain, the R3H domain and the RRM domain. In this research, we investigated the roles of these RNA-binding domains by comparing the structural features and enzymatic properties of mutants lacking either one or two of the three RNA-binding domains. The results showed that the R3H domain had the ability to bind various oligonucleotides at the micromolar level with no oligo(A) specificity. The removal of the R3H domain dissociated PARN into monomers, which still possessed the RNA-binding ability and catalytic functions. Unlike the critical role of the RRM domain in PARN processivity, the removal of the R3H domain did not affect the catalytic pattern of PARN. Our results suggested that both R3H and RRM domains were essential for the high affinity of long poly(A) substrate, but the R3H domain did not contribute to the substrate recognition of PARN. Compared to the RRM domain, the R3H domain played a more important role in the structural integrity of the dimeric PARN. The multiple RNA-binding domain architecture endows PARN the property of highly efficient catalysis in a highly processive mode.

Protein synthesis and degradation gene SNPs related to feed intake, feed efficiency, growth, and ultrasound carcass traits in Nellore cattle

We looked for possible associations of SNPs in genes related to protein turnover, with growth, feed efficiency and carcass traits in feedlot Nellore cattle. Purebred Nellore bulls and steers (N = 290; 378 ± 42 kg body weight, 23 months ± 42 days old) were evaluated for daily feed intake, body weight gain (BWG), gross feed efficiency, feed conversion ratio, partial efficiency of growth, residual feed intake (RFI), ultrasound backfat, rump fat, and ribeye area. Genotypes were obtained for SNPs in the growth hormone receptor (GHR-1 and GHR-2); calpain (CAPN4751); calpastatin (UoGCAST); ubiquitin-conjugating enzyme 2I (UBE2I-1 and UBE2I-2); R3H domain containing 1 (R3HDM1-1, -2, -3, and -4), ring finger protein 19 (RNF19); proteasome 26S subunit, non-ATPase, 13 (PSMD13); ribosomal protein, large, P2 (RPLP2); and isoleucine-tRNA synthetase 2, mitochondrial (IARS2) genes. Allelic substitution, additive and dominant effects were tested and molecular breeding values were computed. CAPN4751, GHR-1 and -2, IARS2, R3HDM1-4, and UoGCAST were found to be normally segregating polymorphisms. Additive and dominance effects were observed on BWG, feed efficiency and carcass traits, although dominant effects predominated. Significant allelic substitution effects were observed for CAPN4751, GHR-1 and -2, and UoGCAST on BWG, gross feed efficiency, RFI, and carcass traits, under single- or multiple-marker analyses. Correlations between molecular breeding values and phenotypes were low, excepted for RFI, based on allelic substitution estimates obtained by stepwise linear regression. We conclude that SNPs in genes related to protein turnover are related to economically important traits in Nellore cattle.

Structural Basis for 5′-End-Specific Recognition of Single-Stranded DNA by the R3H Domain from Human Sμbp-2

The R3H domain is a conserved sequence motif in nucleic acid binding proteins. Previously, we reported the solution structure of the R3H domain and identified a putative nucleic acid binding site composed of three conserved basic residues [Liepinsh, E., Leonchiks, A., Sharipo, A., Guignard, L. & Otting, G. (2003). Solution structure of the R3H domain from human Sμbp-2. J. Mol. Biol.326, 217-223]. Here, we determine the binding affinities of mononucleotides and dinucleotides for the R3H domain from human Sμbp-2 (Sμbp2-R3H) and map their binding sites on the protein's surface. Although the binding affinities show up to 260-fold selectivity between different nucleotides, their binding sites and conformations seem very similar. Further, we report the NMR structure of the Sμbp2-R3H in complex with deoxyguanosine 5'-monophosphate (dGMP) mimicking the 5'-end of single-stranded DNA. Pseudocontact shifts from a paramagnetic lanthanide tag attached to residue 731 in the mutant A731C confirmed that binding of dGMP brings a loop of the protein into closer proximity. The structure provides the first structural insight into single-stranded nucleic acid recognition by the R3H domain and shows that the R3H domain specifically binds the phosphorylated 5'-end through electrostatic interactions with the two conserved arginines and stacking interactions with the highly conserved histidine.

The Ighmbp2 helicase structure reveals the molecular basis for disease-causing mutations in DMSA1

Mutations in immunoglobulin µ-binding protein 2 (Ighmbp2) cause distal spinal muscular atrophy type 1 (DSMA1), an autosomal recessive disease that is clinically characterized by distal limb weakness and respiratory distress. However, despite extensive studies, the mechanism of disease-causing mutations remains elusive. Here we report the crystal structures of the Ighmbp2 helicase core with and without bound RNA. The structures show that the overall fold of Ighmbp2 is very similar to that of Upf1, a key helicase involved in nonsense-mediated mRNA decay. Similar to Upf1, domains 1B and 1C of Ighmbp2 undergo large conformational changes in response to RNA binding, rotating 30° and 10°, respectively. The RNA binding and ATPase activities of Ighmbp2 are further enhanced by the R3H domain, located just downstream of the helicase core. Mapping of the pathogenic mutations of DSMA1 onto the helicase core structure provides a molecular basis for understanding the disease-causing consequences of Ighmbp2 mutations.

Proteomics Identification of Drosophila Small Interfering RNA-associated Factors

The Drosophila melanogaster RNA-induced silencing complex (RISC) forms a large ribonucleoprotein particle on small interfering RNAs (siRNAs) and catalyzes target mRNA cleavage during RNA interference (RNAi). Dicer-2, R2D2, Loquacious, and Argonaute-2 are examples of RISC-associated factors that are involved in RNAi. Holo-RISC is an approximately 80 S small interfering ribonucleoprotein, which suggests that there are many additional proteins that participate in the RNAi pathway. In this study, we used siRNA affinity capture combined with mass spectrometry to identify novel components of the Drosophila RNAi machinery. Our study identified both established RISC components and novel siRNA-associated factors, many of which contain domains that are consistent with potential roles in RNAi. Functional analysis of these novel siRNA-associated proteins suggests that these factors may play an important role in RNAi.

NFX1-123 Increases hTERT Expression and Telomerase Activity Posttranscriptionally in Human Papillomavirus Type 16 E6 Keratinocytes

High-risk human papillomavirus (HPV) E6 protein induces telomerase activity through transcriptional activation of hTERT, the catalytic subunit of telomerase. HPV type 16 (HPV16) E6 interacts with two splice variants of NFX1 to increase hTERT expression. NFX1-91 is a transcriptional repressor of hTERT that is polyubiquitinated and targeted for degradation by HPV16 E6 in concert with E6-associated protein. We previously showed that NFX1-123 augments hTERT expression through binding to cytoplasmic poly(A) binding proteins (PABPCs). In this study, we determined that unlike NFX1-91, NFX1-123 is a cytoplasmic protein that colocalized with PABPCs but does not shuttle with PABPCs between the nucleus and cytoplasm. NFX1-123 requires both its PAM2 motif, with which it binds PABPCs, and its R3H domain, which has putative nucleic acid binding capabilities, to increase hTERT mRNA levels and telomerase activity in keratinocytes expressing HPV16 E6. In keratinocytes expressing HPV16 E6 and overexpressing NFX1-123, there was increased protein expression from in vitro-transcribed RNA fused with the 5' untranslated region (5' UTR) of hTERT. This posttranscriptional increase in expression required the PAM2 motif and R3H domain of NFX1-123 as well as the coexpression of HPV16 E6. NFX1-123 bound endogenous hTERT mRNA and increased its stability in HPV16 E6-expressing human foreskin keratinocytes, and NFX1-123 increased the stability of in vitro-transcribed RNA fused with the 5' UTR of hTERT. Together, these studies describe the first evidence of posttranscriptional regulation of hTERT, through the direct interaction of the cytoplasmic protein NFX1-123 with hTERT mRNA, in HPV16 E6-expressing keratinocytes.

Structural Basis of m7GpppG Binding to Poly(A)-Specific Ribonuclease

Poly(A)-specific ribonuclease (PARN) is a homodimeric, processive, and cap-interacting 3' exoribonuclease that efficiently degrades eukaryotic mRNA poly(A) tails. The crystal structure of a C-terminally truncated PARN in complex with m(7)GpppG reveals that, in one subunit, m(7)GpppG binds to a cavity formed by the RRM domain and the nuclease domain, whereas in the other subunit, it binds almost exclusively to the RRM domain. Importantly, our structural and competition data show that the cap-binding site overlaps with the active site in the nuclease domain. Mutational analysis demonstrates that residues involved in m(7)G recognition are crucial for cap-stimulated deadenylation activity, and those involved in both cap and poly(A) binding are important for catalysis. A modeled PARN, which shows that the RRM domain from one subunit and the R3H domain from the other subunit enclose the active site, provides a structural foundation for further studies to elucidate the mechanism of PARN-mediated deadenylation.

The CAP Superfamily: Cysteine-Rich Secretory Proteins, Antigen 5, and Pathogenesis-Related 1 Proteins—Roles in Reproduction, Cancer, and Immune Defense

The cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP) superfamily members are found in a remarkable range of organisms spanning each of the animal kingdoms. Within humans and mice, there are 31 and 33 individual family members, respectively, and although many are poorly characterized, the majority show a notable expression bias to the reproductive tract and immune tissues or are deregulated in cancers. CAP superfamily proteins are most often secreted and have an extracellular endocrine or paracrine function and are involved in processes including the regulation of extracellular matrix and branching morphogenesis, potentially as either proteases or protease inhibitors; in ion channel regulation in fertility; as tumor suppressor or prooncogenic genes in tissues including the prostate; and in cell-cell adhesion during fertilization. This review describes mammalian CAP superfamily gene expression profiles, phylogenetic relationships, protein structural properties, and biological functions, and it draws into focus their potential role in health and disease. The nine subfamilies of the mammalian CAP superfamily include: the human glioma pathogenesis-related 1 (GLIPR1), Golgi associated pathogenesis related-1 (GAPR1) proteins, peptidase inhibitor 15 (PI15), peptidase inhibitor 16 (PI16), cysteine-rich secretory proteins (CRISPs), CRISP LCCL domain containing 1 (CRISPLD1), CRISP LCCL domain containing 2 (CRISPLD2), mannose receptor like and the R3H domain containing like proteins. We conclude that overall protein structural conservation within the CAP superfamily results in fundamentally similar functions for the CAP domain in all members, yet the diversity outside of this core region dramatically alters target specificity and, therefore, the biological consequences.