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RNA–protein interaction networks govern many biological processes but are difficult to examine comprehensively. We devised ribonucleoprotein networks analyzed by mutational profiling (RNP-MaP), a live-cell chemical probing strategy that maps cooperative interactions among multiple proteins bound to single RNA molecules at nucleotide resolution. RNP-MaP uses a hetero-bifunctional crosslinker to freeze interacting proteins in place on RNA and then maps multiple bound proteins on single RNA strands by read-through reverse transcription and DNA sequencing. RNP-MaP revealed that RNase P and RMRP, two sequence-divergent but structurally related non-coding RNAs, share RNP networks and that network hubs define functional sites in these RNAs. RNP-MaP also identified protein interaction networks conserved between mouse and human XIST long non-coding RNAs and defined protein communities whose binding sites colocalize and form networks in functional regions of XIST. RNP-MaP enables discovery and efficient validation of functional protein interaction networks on long RNAs in living cells. Networks of proteins bound to single RNAs are identified by correlated chemical crosslinking.

Non-small cell lung cancer (NSCLC) has the highest mortality rate among all malignancies worldwide. The role of long noncoding RNAs (lncRNAs) in the progression of cancers is a contemporary research hotspot. Based on an integrative analysis of The Cancer Genome Atlas database, we identified lncRNA-RNA Component of Mitochondrial RNA Processing Endoribonuclease (RMRP) as one of the most highly upregulated lncRNAs that are associated with poor survival in NSCLC. Furthermore, N(6)-methyladenosine (m6A) was highly enriched within RMRP and enhanced its RNA stability. In vitro and in vivo experiments showed that RMRP promoted NSCLC cell proliferation, invasion, and migration. In terms of mechanism, RMRP recruited YBX1 to the TGFBR1 promotor region, leading to upregulation of the transcription of TGFBR1. The TGFBR1/SMAD2/SMAD3 pathway was also regulated by RMRP. In addition, RMRP promoted the cancer stem cells properties and epithelial mesenchymal transition, which promote the resistance to radiation therapy and cisplatin. Clinical data further confirmed a positive correlation between RMRP and TGFBR1. In short, our work reveals that m6A RNA methylation-mediated RMRP stability renders proliferation and progression of NSCLC through regulating TGFBR1/SMAD2/SMAD3 pathway.

Genomic analysis of tumours has led to the identification of hundreds of cancer genes on the basis of the presence of mutations in protein-coding regions. By contrast, much less is known about cancer-causing mutations in non-coding regions. Here we perform deep sequencing in 360 primary breast cancers and develop computational methods to identify significantly mutated promoters. Clear signals are found in the promoters of three genes. FOXA1 , a known driver of hormone-receptor positive breast cancer, harbours a mutational hotspot in its promoter leading to overexpression through increased E2F binding. RMRP and NEAT1 , two non-coding RNA genes, carry mutations that affect protein binding to their promoters and alter expression levels. Our study shows that promoter regions harbour recurrent mutations in cancer with functional consequences and that the mutations occur at similar frequencies as in coding regions. Power analyses indicate that more such regions remain to be discovered through deep sequencing of adequately sized cohorts of patients. High-depth sequencing of targeted regions in primary breast cancer identifies mutated promoter elements with recurrent mutations at specific and/or nearby bases, suggesting selection of certain non-coding events. Non-coding driver mutations for breast cancer Many cancer genomic studies have characterized the landscape of driver mutations in protein-coding regions, but there has been limited exploration of non-coding regions. Gad Getz and colleagues searched for significantly mutated regulatory regions with high-depth sequencing of targeted regions in 360 primary breast cancers. They identified significantly mutated promoter elements associated with nine genes, and found recurrent mutations at specific and/or nearby bases, suggesting targeting of a specific element. For three of these genes, FOXA1 , RMRP and NEAT1 , they show that these recurrent promoter-proximal mutations influence gene expression and protein binding affinity. The authors suggest that further deep sequencing studies in larger cohorts could identify more as yet undiscovered promoter regions.

RNA structure and dynamics are critical to biological function. However, strategies for determining RNA structure in vivo are limited, with established chemical probing and newer duplex detection methods each having deficiencies. Here we convert the common reagent dimethyl sulfate into a useful probe of all 4 RNA nucleotides. Building on this advance, we introduce PAIR-MaP, which uses single-molecule correlated chemical probing to directly detect base-pairing interactions in cells. PAIR-MaP has superior resolution compared to alternative experiments, can resolve multiple sets of pairing interactions for structurally dynamic RNAs, and enables highly accurate structure modeling, including of RNAs containing multiple pseudoknots and extensively bound by proteins. Application of PAIR-MaP to human RNase MRP and 2 bacterial messenger RNA 5′ untranslated regions reveals functionally important and complex structures undetected by prior analyses. PAIR-MaP is a powerful, experimentally concise, and broadly applicable strategy for directly visualizing RNA base pairs and dynamics in cells.

RNase P and MRP are highly conserved, multi-protein/RNA complexes with essential roles in processing ribosomal and tRNAs. Three proteins found in both complexes, Pop1, Pop6, and Pop7 are also telomerase-associated. Here, we determine how temperature sensitive POP1 and POP6 alleles affect yeast telomerase. At permissive temperatures, mutant Pop1/6 have little or no effect on cell growth, global protein levels, the abundance of Est1 and Est2 (telomerase proteins), and the processing of TLC1 (telomerase RNA). However, in pop mutants, TLC1 is more abundant, telomeres are short, and TLC1 accumulates in the cytoplasm. Although Est1/2 binding to TLC1 occurs at normal levels, Est1 (and hence Est3) binding is highly unstable. We propose that Pop-mediated stabilization of Est1 binding to TLC1 is a pre-requisite for formation and nuclear localization of the telomerase holoenzyme. Furthermore, Pop proteins affect TLC1 and the RNA subunits of RNase P/MRP in very different ways. Pop1 and 6 are subunits of RNase P and RNase MRP, which process ribosomal and tRNAs. The authors show that when Pop1 and 6 are impaired, the telomerase subunit Est1 binds telomerase RNA at normal levels, but the binding is unstable. As a result, nuclear import of the telomerase holoenzyme is inhibited.

p53 inactivation is highly associated with tumorigenesis and drug resistance. Here, we identify a long noncoding RNA, the RNA component of mitochondrial RNA-processing endoribonuclease (RMRP), as an inhibitor of p53. RMRP is overexpressed and associated with an unfavorable prognosis in colorectal cancer. Ectopic RMRP suppresses p53 activity by promoting MDM2-induced p53 ubiquitination and degradation, while depletion of RMRP activates the p53 pathway. RMRP also promotes colorectal cancer growth and proliferation in a p53-dependent fashion in vitro and in vivo. This anti-p53 action of RMRP is executed through an identified partner protein, SNRPA1. RMRP can interact with SNRPA1 and sequester it in the nucleus, consequently blocking its lysosomal proteolysis via chaperone-mediated autophagy. The nuclear SNRPA1 then interacts with p53 and enhances MDM2-induced proteasomal degradation of p53. Remarkably, ablation of SNRPA1 completely abrogates RMRP regulation of p53 and tumor cell growth, indicating that SNRPA1 is indispensable for the anti-p53 function of RMRP. Interestingly and significantly, poly (ADP-ribose) polymerase (PARP) inhibitors induce RMRP expression through the transcription factor C/EBPβ, and RMRP confers tumor resistance to PARP inhibition by preventing p53 activation. Altogether, our study demonstrates that RMRP plays an oncogenic role by inactivating p53 via SNRPA1 in colorectal cancer.

RNase MRP is an essential eukaryotic ribonucleoprotein complex involved in the maturation of rRNA and the regulation of the cell cycle. RNase MRP is related to the ribozyme-based RNase P, but it has evolved to have distinct cellular roles. We report a cryo-EM structure of the S. cerevisiae RNase MRP holoenzyme solved to 3.0 Å. We describe the structure of this 450 kDa complex, interactions between its components, and the organization of its catalytic RNA. We show that some of the RNase MRP proteins shared with RNase P undergo an unexpected RNA-driven remodeling that allows them to bind to divergent RNAs. Further, we reveal how this RNA-driven protein remodeling, acting together with the introduction of new auxiliary elements, results in the functional diversification of RNase MRP and its progenitor, RNase P, and demonstrate structural underpinnings of the acquisition of new functions by catalytic RNPs. Ribozyme-based RNase MRP is an essential eukaryotic enzyme involved in the maturation of rRNA and is evolutionarily related to RNase P. Here, the authors present the 3.0 Å cryo-EM structure of the S. cerevisiae RNase MRP holoenzyme, a 450 kDa ribonucleoprotein complex and compare it with RNase P.

RNA binding proteins (RBPs) play pivotal roles in breast cancer (BC) development. As an RBP, Processing of precursor 7 (POP7) is one of the subunits of RNase P and RNase MRP, however, its exact function and mechanism in BC remain unknown. Here, we showed that expression of POP7 was frequently increased in BC cells and in primary breast tumors. Upregulated POP7 significantly promoted BC cell proliferation in vitro and primary tumor growth in vivo. POP7 also increased cell migration, invasion in vitro, and lung metastasis in vivo. Through RNA immunoprecipitation coupled with sequencing (RIP‐seq), we found that POP7 bound preferentially to intron regions and POP7‐binding peak associated genes were mainly enriched in cancer‐related pathways. Furthermore, POP7 regulated Interleukin Enhancer Binding Factor 3 (ILF3) expression through influencing its mRNA stability. Knockdown of ILF3 significantly impaired the increased malignant potential of POP7‐overexpressing cells, suggesting that POP7 enhances BC progression through regulating ILF3 expression. Collectively, our findings provide the first evidence for the important role of POP7 and its regulation of ILF3 in promoting BC progression. Our findings provide the first evidence for RNA‐binding protein POP7 in promoting breast cancer progression. Through RIP‐seq, we found that POP7 bound preferentially to intron regions of target RNAs and ILF3 was identified as one of the POP7‐binding transcripts. And regulation of ILF3 expression and mRNA stability is through which POP7 exerts its function on promoting breast cancer.

The DEDD family of exonucleases has expanded through evolution whilst retaining a conserved catalytic domain. One subgroup with closely related catalytic DEDD domain sequences includes the yeast enzymes Rex1 (RNA exonuclease 1) and Rex3, the metazoan REXO1 (RNA exonuclease 1 homologue) and Rexo5 proteins, and the plant protein Sdn5 (small RNA degrading nuclease). Comparison of protein structure models and sequence analyses revealed that this group can be differentiated into two distinct clades consisting of Rex1, Rexo5 and Sdn5 on the one hand, and Rex3 and REXO1 on the other. The catalytic domain of Rex1-related proteins is inserted within a conserved, discontinuous alkaline phosphatase (AlkP) domain. The AlkP domain of yeast Rex1 contains three surface loops that are modelled to be directed towards the DEDD domain, one of which forms an extended helical arch that is found in homologues across fungi and plants. We show that this arch and an adjacent loop are required for Rex1-mediated processing of 5S rRNA and tRNA in Saccharomyces cerevisiae . Rex3-related proteins, including REXO1, lack the AlkP domain but contain a KIX domain (CREB kinase-inducible domain (KID) interacting domain) and a cysteine- and histidine-rich domain (CHORD) adjacent to a C-terminal DEDD domain. Deletion of the N-terminal region within yeast Rex3 spanning the KIX domain blocked its function in RNase MRP processing. In contrast to Rex1, Rex3 proteins are found in metazoans and fungi but not in plants or algae. This work identifies evolutionarily conserved structural hallmarks within Rex1 and Rex3 proteins and demonstrates that specific features are required for Rex1- and Rex3-mediated RNA processing pathways in vivo.

BACKGROUND: T-cell receptor excision circles (TREC) and κ-deleting recombination receptor excision circles (KREC) concentrations can be used to assess and diagnose immune deficiencies, monitor thymic and bone marrow immune reconstitution, or follow responses to drug therapy. We developed an assay to quantify TREC, KREC, and a reference gene in a single reaction using droplet digital PCR (ddPCR). METHODS: PCR was optimized for 3 targets: TREC, KREC, and ribonuclease P/MRP subunit p30 (RPP30) as the reference gene. Multiplexing was accomplished by varying the target's fluorophore and concentration. Correlation with clinical results was evaluated using 47 samples from healthy donors, 59 samples with T-cell and B-cell markers within the reference interval from the flow cytometry laboratory, 20 cord blood samples, and 34 samples submitted for exome sequencing for severe combined immunodeficiency disease (SCID). RESULTS: The limit of the blank was 4 positive droplets, limit of detection 9 positive droplets, and limit of quantification 25 positive droplets, or 2.0 copies/µL. TREC and KREC copies/µL were as expected in the healthy donors and cord blood samples and concordant with the healthy flow cytometry results. Of the samples from the SCID Panel, 56.5% had a TREC count<20 copies/µL and 17.7% had a KREC count<20 copies/µL, suggestive of low T- and B-cell numbers, respectively. CONCLUSIONS: Our multiplex ddPCR assay is an analytically sensitive and specific method for the absolute quantification of TREC and KREC. To the best of our knowledge, this paper is the first to describe the simultaneous quantification of TREC, KREC, and a reference gene by use of ddPCR.