RNA Interactions Research Articles

RUNX1 C-terminal mutations impair blood cell differentiation by perturbing specific enhancer-promoter networks

AbstractThe transcription factor RUNX1 is a master regulator of hematopoiesis and is frequently mutated in myeloid malignancies. Mutations in its runt homology domain (RHD) frequently disrupt DNA binding and result in loss of RUNX1 function. However, it is not clearly understood how other RUNX1 mutations contribute to disease development. Here, we characterized RUNX1 mutations outside of the RHD. Our analysis of the patient data sets revealed that mutations within the C-terminus frequently occur in hematopoietic disorders. Remarkably, most of these mutations were nonsense or frameshift mutations and were predicted to be exempt from nonsense-mediated messenger RNA decay. Therefore, this class of mutation is projected to produce DNA-binding proteins that contribute to the pathogenesis in a distinct manner. To model this, we introduced the RUNX1R320∗ mutation into the endogenous gene locus and demonstrated the production of RUNX1R320∗ protein. Expression of RUNX1R320∗ resulted in the disruption of RUNX1 regulated processes such as megakaryocytic differentiation, through a transcriptional signature different from RUNX1 depletion. To understand the underlying mechanisms, we used Global RNA Interactions with DNA by deep sequencing (GRID-seq) to examine enhancer-promoter connections. We identified widespread alterations in the enhancer-promoter networks within RUNX1 mutant cells. Additionally, we uncovered enrichment of RUNX1R320∗ and FOXK2 binding at the MYC super enhancer locus, significantly upregulating MYC transcription and signaling pathways. Together, our study demonstrated that most RUNX1 mutations outside the DNA-binding domain are not subject to nonsense-mediated decay, producing protein products that act in concert with additional cofactors to dysregulate hematopoiesis through mechanisms distinct from those induced by RUNX1 depletion.

Regulation of inflammatory response by LINC00346 via miR-25-3p-mediated modulation of the PTEN/PI3K/AKT/NF-κB pathway

Long intergenic non-coding RNA 346 (LINC00346) has been reported to be involved in the development of atherosclerosis and specific cancers by affecting signaling pathways. However, its function in inflammation has not been thoroughly studied. Therefore, its expression pattern and function were determined in the human macrophage-like cell line THP-1. Lipopolysaccharide (LPS) treatment induced the expression of LINC00346. LPS-induced NF-κB activation and proinflammatory cytokine expression were suppressed or enhanced by the overexpression or knockdown of LINC00346, respectively. Analyses using dual luciferase assay and decoy RNAs that could block RNA–RNA interactions indicated that LINC00346 improves phosphatase and tensin homolog (PTEN) expression by sponging miR-25–3p. Subsequently, PTEN suppresses phosphoinositide-3 kinase (PI3K)-mediated conversion of phosphatidylinositol-4,5-bisphosphate (PIP2) into phosphatidylinositol-3,4,5-trisphosphate (PIP3) as well as consequent activation of protein kinase B (AKT) and NF-κB. Interestingly, database analysis revealed that the expression levels of LINC00346 and PTEN were simultaneously decreased in breast cancer tissues. Further analyses conducted using a breast cancer cell line, MDA-MB-231, confirmed the functional relationship among LINC00346, miR-25–3p, and PTEN in LPS-induced activation of NF-κB. These results indicate that miR-25-3p-sponging activity of LINC00346 affects the balance between PTEN and PI3K as well as the downstream activation of AKT/NF-κB pathway in inflammatory conditions.

Abstract 4386: Androgen hormone signaling regulates the association between the hippo pathway component YAP1 and RNA binding protein NPM1

Abstract Prostate cancer represents a major health concern for men in the United States and globally, disproportionately affecting men of African descent. Despite recent advances, the precise etiology and molecular mechanisms underpinning fatal prostate cancer in humans remain inadequately understood. RNA binding proteins (RBPs) regulate various aspects of RNA biology, and when their regulation is disrupted, they can lead to human diseases, including cancer. However, the comprehensive alterations in RBP-mRNA interactions in prostate cancer have not been explored. In this study, we investigated the dynamics of RBPs and their interactions with polyadenylated mRNA in the castration-sensitive LNCaP and castration-resistant C4-2 prostate cancer cell lines. Using an enhanced RNA interactome capture assay in combination with mass spectrometry, we discovered that LNCaP and C4-2 cells showed distinct RBP-mRNA interaction patterns in steady-state growth conditions and in response to androgen treatment. Additionally, we investigated the interplay between the oncogene Yes-associated protein 1 (YAP1) and the RNA-binding protein nucleophosmin (NPM1). We identified NPM1 as a component of the YAP1 protein complex through mass spectrometry-based proteomics. Our proximity ligation assay demonstrated that androgen signaling could regulate the interaction between YAP1 and NPM1. Also, our GST-pulldown revealed that NPM1 binds to the proline-rich domain of YAP1 in LNCaP cells, while the WW/SH3 domain of YAP1 mediates the interaction with NPM1 in C4-2 cells. Genetic and pharmacological inhibition of NPM1 activity reduced prostate cancer cell growth in culture by modulating cell cycle progression. Furthermore, our computational and immunological analysis of prostate tumor tissues indicated that the upregulation of NPM1 might be associated with the clinical advancement of human prostate cancer. These findings underscore the critical role of the molecular functional connection between YAP1 and RBPs and the global changes in RBP and RNA interaction patterns, in the progression and recurrence of prostate cancer. Citation Format: Abdulrahman M. Dwead, Marwah M. Al-Mathkour, Bekir Cinar. Androgen hormone signaling regulates the association between the hippo pathway component YAP1 and RNA binding protein NPM1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4386.

Txtools: an R package facilitating analysis of RNA modifications, structures, and interactions.

We present txtools, an R package that enables the processing, analysis, and visualization of RNA-seq data at the nucleotide-level resolution, seamlessly integrating alignments to the genome with transcriptomic representation. txtools' main inputs are BAM files and a transcriptome annotation, and the main output is a table, capturing mismatches, deletions, and the number of reads beginning and ending at each nucleotide in the transcriptomic space. txtools further facilitates downstream visualization and analyses. We showcase, using examples from the epitranscriptomic field, how a few calls to txtools functions can yield insightful and ready-to-publish results. txtools is of broad utility also in the context of structural mapping and RNA:protein interaction mapping. By providing a simple and intuitive framework, we believe that txtools will be a useful and convenient tool and pave the path for future discovery. txtools is available for installation from its GitHub repository at https://github.com/AngelCampos/txtools.

RNA modulates hnRNPA1A amyloid formation mediated by biomolecular condensates

Several RNA binding proteins involved in membraneless organelles can form pathological amyloids associated with neurodegenerative diseases, but the mechanisms of how this aggregation is modulated remain elusive. Here we investigate how heterotypic protein–RNA interactions modulate the condensation and the liquid to amyloid transition of hnRNPA1A, a protein involved in amyothropic lateral sclerosis. In the absence of RNA, formation of condensates promotes hnRNPA1A aggregation and fibrils are localized at the interface of the condensates. Addition of RNA modulates the soluble to amyloid transition of hnRNPA1A according to different pathways depending on RNA/protein stoichiometry. At low RNA concentrations, RNA promotes both condensation and amyloid formation, and the catalytic effect of RNA adds to the role of the interface between the dense and dilute phases. At higher RNA concentrations, condensation is suppressed according to re-entrant phase behaviour but formation of hnRNPA1A amyloids is observed over longer incubation times. Our findings show how heterotypic nucleic acid–protein interactions affect the kinetics and molecular pathways of amyloid formation.

Role and regulation of FOXO3a: new insights into breast cancer therapy.

Breast cancer is the most common malignancy in the world, particularly affecting female cancer patients. Enhancing the therapeutic strategies for breast cancer necessitates identifying molecular drug targets that effectively eliminate tumor cells. One of these prominent targets is the forkhead and O3a class (FOXO3a), a member of the forkhead transcription factor subfamily. FOXO3a plays a pivotal role in various cellular processes, including apoptosis, proliferation, cell cycle regulation, and drug resistance. It acts as a tumor suppressor in multiple cancer types, although its specific role in cancer remains unclear. Moreover, FOXO3a shows promise as a potential marker for tumor diagnosis and prognosis in breast cancer patients. In addition, it is actively influenced by common anti-breast cancer drugs like paclitaxel, simvastatin, and gefitinib. In breast cancer, the regulation of FOXO3a involves intricate networks, encompassing post-translational modification post-translational regulation by non-coding RNA (ncRNA) and protein-protein interaction. The specific mechanism of FOXO3a in breast cancer urgently requires further investigation. This review aims to systematically elucidate the role of FOXO3a in breast cancer. Additionally, it reviews the interaction of FOXO3a and its upstream and downstream signaling pathway-related molecules to uncover potential therapeutic drugs and related regulatory factors for breast cancer treatment by regulating FOXO3a.

To initiate or not to initiate: A critical assessment of eIF2A, eIF2D, and MCT-1·DENR to deliver initiator tRNA to ribosomes.

Selection of the correct start codon is critical for high-fidelity protein synthesis. In eukaryotes, this is typically governed by a multitude of initiation factors (eIFs), including eIF2·GTP that directly delivers the initiator tRNA (Met-tRNAi Met ) to the P site of the ribosome. However, numerous reports, some dating back to the early 1970s, have described other initiation factors having high affinity for the initiator tRNA and the ability of delivering it to the ribosome, which has provided a foundation for further work demonstrating non-canonical initiation mechanisms using alternative initiation factors. Here we provide a critical analysis of current understanding of eIF2A, eIF2D, and the MCT-1·DENR dimer, the evidence surrounding their ability to initiate translation, their implications in human disease, and lay out important key questions for the field. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Translation > Mechanisms Translation > Regulation.

Beyond reader proteins: RNA binding proteins and RNA modifications in conversation to regulate gene expression.

Post-transcriptional mRNA modifications play diverse roles in gene expression and RNA function. In many cases, RNA modifications function by altering how cellular machinery such as RNA binding proteins (RBPs) interact with RNA substrates. For instance, N6-methyladenosine (m6A) is recognized by the well-characterized YTH domain-containing family of "reader" proteins. For other mRNA modifications, similar global readers of modification status have not been clearly defined. Rather, most interactions between RBPs and RNA modifications have a more complicated dependence on sequence context and binding modality. The current handful of studies that demonstrate modifications impacting protein binding likely represent only a fraction of the full landscape. In this review, we dissect the known instances of RNA modifications altering RBP binding, specifically m6A, N1-methyladenosine (m1A), 5-methylcytosine (m5C), pseudouridine (Ψ), and internal N7-methylguanosine. We then review the biochemical properties of these and other identified mRNA modifications including dihydrouridine (D), N4-acetylcytosine (ac4C), and 2'-O-Methylation (Nme). We focus on how these properties would be likely to impact RNA:RBP interactions, including by changes to hydrogen bond potential, base-stacking efficiency, and RNA conformational preferences. The effects of RNA modifications on secondary structure have been well-studied, and we briefly discuss how structural effects imparted by modifications can lead to protein binding changes. Finally, we discuss strategies for uncovering as-yet-to-be identified modification-sensitive RBP:RNA Interactions. Coordinating future efforts to intersect the epitranscriptome and the RNA-protein interactome will illuminate the rules governing RNA modification recognition and the mechanisms responsible for the biological consequences of mRNA modification. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Processing > RNA Editing and Modification.

RNA-binding proteins in pain.

RNAs are meticulously controlled by proteins. Through direct and indirect associations, every facet in the brief life of an mRNA is subject to regulation. RNA-binding proteins (RBPs) permeate biology. Here, we focus on their roles in pain. Chronic pain is among the largest challenges facing medicine and requires new strategies. Mounting pharmacologic and genetic evidence obtained in pre-clinical models suggests fundamental roles for a broad array of RBPs. We describe their diverse roles that span RNA modification, splicing, stability, translation, and decay. Finally, we highlight opportunities to expand our understanding of regulatory interactions that contribute to pain signaling. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications Translation > Regulation RNA in Disease and Development > RNA in Disease.

Interaction of Long Non-Coding RNAs and MicroRNAs on Human Papillomavirus and Epstein–Barr Virus-Associated Cancers: A Narrative Review

Interaction of Long Non-Coding RNAs and MicroRNAs on Human Papillomavirus and Epstein–Barr Virus-Associated Cancers: A Narrative Review

Stoichiometry of long noncoding RNA interactions with other RNAs: Insights from OIP5-AS1.

Long noncoding (lnc)RNAs modulate gene expression programs in a range of developmental processes in different organs. In skeletal muscle, lncRNAs have been implicated in myogenesis, the process whereby muscle precursor cells form muscle fibers during embryonic development and regenerate muscle fibers in the adult. Here, we discuss OIP5-AS1, a lncRNA that is highly expressed in skeletal muscle and is capable of coordinating protein expression programs during myogenesis. Given that several myogenic functions of OIP5-AS1 involve interactions with MEF2C mRNA and with the microRNA miR-7, it was critical to carefully evaluate the precise levels of OIP5-AS1 during myogenesis. We discuss the approaches used to examine lncRNA copy number using OIP5-AS1 as an example, focusing on quantification by quantitative PCR analysis with reference to nucleic acids of known abundance, by droplet digital (dd)PCR measurement, and by microscopic visualization of individual lncRNAs in cells. We discuss considerations of RNA stoichiometry in light of developmental processes in which lncRNAs are implicated. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

Multiple structural flavors of RNase P in precursor tRNA processing.

The precursor transfer RNAs (pre-tRNAs) require extensive processing to generate mature tRNAs possessing proper fold, structural stability, and functionality required to sustain cellular viability. The road to tRNA maturation follows an ordered process: 5'-processing, 3'-processing, modifications at specific sites, if any, and 3'-CCA addition before aminoacylation and recruitment to the cellular protein synthesis machinery. Ribonuclease P (RNase P) is a universally conserved endonuclease in all domains of life, performing the hydrolysis of pre-tRNA sequences at the 5' end by the removal of phosphodiester linkages between nucleotides at position -1 and +1. Except for an archaeal species: Nanoarchaeum equitans where tRNAs are transcribed from leaderless-position +1, RNase P is indispensable for life and displays fundamental variations in terms of enzyme subunit composition, mechanism of substrate recognition and active site architecture, utilizing in all cases a two metal ion-mediated conserved catalytic reaction. While the canonical RNA-based ribonucleoprotein RNase P has been well-known to occur in bacteria, archaea, and eukaryotes, the occurrence of RNA-free protein-only RNase P in eukaryotes and RNA-free homologs of Aquifex RNase P in prokaryotes has been discovered more recently. This review aims to provide a comprehensive overview of structural diversity displayed by various RNA-based and RNA-free RNase P holoenzymes towards harnessing critical RNA-protein and protein-protein interactions in achieving conserved pre-tRNA processing functionality. Furthermore, alternate roles and functional interchangeability of RNase P are discussed in the context of its employability in several clinical and biotechnological applications. This article is categorized under: RNA Processing > tRNA Processing RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

Cryo-EM structure of rotavirus B NSP2 reveals its unique tertiary architecture.

Rotavirus (RV) NSP2 is a multifunctional RNA chaperone that exhibits numerous activities that are essential for replication and viral genome packaging. We performed an in silico analysis that highlighted a distant relationship of NSP2 from rotavirus B (RVB) to proteins from other human RVs. We solved a cryo-electron microscopy structure of RVB NSP2 that shows structural differences with corresponding proteins from other human RVs. Based on the structure, we identified amino acid residues that are involved in RNA interactions. Anisotropy titration experiments showed that these residues are important for nucleic acid binding. We also identified structural motifs that are conserved in all RV species. Collectively, our data complete the structural characterization of rotaviral NSP2 protein and demonstrate its structural diversity among RV species.IMPORTANCERotavirus B (RVB), also known as adult diarrhea rotavirus, has caused epidemics of severe diarrhea in China, India, and Bangladesh. Thousands of people are infected in a single RVB epidemic. However, information on this group of rotaviruses remains limited. As NSP2 is an essential protein in the viral life cycle, including its role in the formation of replication factories, it may be a target for future antiviral strategy against viruses with similar mechanisms.

Bone mesenchymal stem cell-derived exosomal miR-26a-3p promotes autophagy to attenuate LPS-induced apoptosis and inflammation in pulmonary microvascular endothelial cells.

Acute lung injury (ALI) is a serious lung disease. The apoptosis and inflammation of pulmonary microvascular endothelial cells (PMVECs) are the primary reasons for ALI. This study aimed to explore the treatment effect and regulatory mechanism of bone mesenchymal stem cell-derived exosomes (BMSC-expos) on ALI. PMVECs were stimulated by Lipopolysaccharide (LPS) to imitate ALI environment. Cell viability was determined by CCK-8 assay. Cell apoptosis was evaluated by TUNEL and flow cytometry. ELISA was utilized for testing the contents of TNF-α, IL-1β, IL-6, and IL-17. Western blot was applied for testing the levels of autophagy-related proteins LC3, p62, and Beclin-1. RNA interaction was determined by luciferase reporter assay. The ALI rat model was established by intratracheal injection of LPS. Evans blue staining was utilized for detecting pulmonary vascular permeability. Our results showed that LPS stimulation notably reduced cell viability, increased cell apoptosis rate, and enhanced the contents of inflammatory factors in PMVECs. However, BMSC-exo treatment significantly abolished the promoting effects of LPS on cell injury. In addition, we discovered that BMSC-exo treatment notably activated autophagy in LPS-induced PMVECs. Furthermore, BMSC-expos upregulated miR-26a-3p expression and downregulated PTEN in PMVECs. MiR-26a-3p was directly bound to PTEN. MiR-26a-3p overexpression reduced cell apoptosis, and inflammation and promoted autophagy by silencing PTEN. Animal experiments proved that miR-26a-3p overexpression effectively improved LPS-induced lung injury in rats. The results proved that BMSC-expos promotes autophagy to attenuate LPS-induced apoptosis and inflammation in pulmonary microvascular endothelial cells via miR-26a-3p/PTEN axis.

HIV-1 RNA genome packaging: it's G-rated.

A member of the Retroviridae, human immunodeficiency virus type 1 (HIV-1), uses the RNA genome packaged into nascent virions to transfer genetic information to its progeny. The genome packaging step is a highly regulated and extremely efficient process as a vast majority of virus particles contain two copies of full-length unspliced HIV-1 RNA that form a dimer. Thus, during virus assembly HIV-1 can identify and selectively encapsidate HIV-1 unspliced RNA from an abundant pool of cellular RNAs and various spliced HIV-1 RNAs. Several "G" features facilitate the packaging of a dimeric RNA genome. The viral polyprotein Gag orchestrates virus assembly and mediates RNA genome packaging. During this process, Gag preferentially binds unpaired guanosines within the highly structured 5' untranslated region (UTR) of HIV-1 RNA. In addition, the HIV-1 unspliced RNA provides a scaffold that promotes Gag:Gag interactions and virus assembly, thereby ensuring its packaging. Intriguingly, recent studies have shown that the use of different guanosines at the junction of U3 and R as transcription start sites results in HIV-1 unspliced RNA species with 99.9% identical sequences but dramatically distinct 5' UTR conformations. Consequently, one species of unspliced RNA is preferentially packaged over other nearly identical RNAs. These studies reveal how conformations affect the functions of HIV-1 RNA elements and the complex regulation of HIV-1 replication. In this review, we summarize cis- and trans-acting elements critical for HIV-1 RNA packaging, locations of Gag:RNA interactions that mediate genome encapsidation, and the effects of transcription start sites on the structure and packaging of HIV-1 RNA.

UPF1 ATPase autoinhibition and activation modulate RNA binding kinetics and NMD efficiency.

The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited 'closed' state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active 'open' state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants (i.e. poorly processive, slow, and mechanochemically uncoupled) can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower RNA binding kinetics and enhanced ATP-stimulated RNA dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2 and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.

Circ_0007445 inhibits trophoblast cell proliferation, migration and invasion by mediating the miR-4432/HTRA1 axis in preeclampsia.

: Circular RNAs (circRNAs) have been shown to be extensively involved in preeclampsia progression. At present, the role of circ_0007445 in preeclampsia progression is not clear. A total of 30 preeclampsia patients and 30 normal pregnant women were recruited in our study. The function of trophoblast cells was explored to clarify the role and mechanism of circ_0007445 on the preeclampsia progression. The expression of circ_0007445, microRNA (miR)-4432 and high temperature requirement A1 (HTRA1) was analyzed by quantitative real-time PCR. The proliferation, migration and invasion of trophoblast cells were determined by cell counting kit 8 assay, EdU assay, colony formation assay, flow cytometry, and transwell assay. Protein expression was examined by western blot analysis. Dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull-down assay were used to assess RNA interaction relationships. Our data suggested that circ_0007445 had increased expression in preeclampsia patients. Knockdown of circ_0007445 enhanced trophoblast cell proliferation, migration and invasion. MiR-4432 was lowly expressed in preeclampsia patients, and it could be sponged by circ_0007445. MiR-4432 inhibitor overturned the promotion effects of circ_0007445 knockdown on trophoblast cell functions. HTRA1 was highly expressed in preeclampsia patients, and it could be targeted by miR-4432. HTRA1 overexpression could also reverse the proliferation, migration and invasion of trophoblast cells promoted by miR-4432 mimic. In addition, circ_0007445 positively regulated HTRA1 through targeting miR-4432. :Our results suggested that circ_0007445 facilitated the development of preeclampsia by suppressing trophoblast cell function through miR-4432/HTRA1 axis.

LncRNA-miRNA-mRNA network in kidney transcriptome of Labeo rohita under hypersaline environment

The present study describes the kidney transcriptome of Labeo rohita, a freshwater fish, exposed to gradually increased salinity concentrations (2, 4, 6 and 8ppt). A total of 10.25 Gbps data was generated, and a suite of bioinformatics tools, including FEELnc, CPC2 and BLASTn were employed for identification of long non-coding RNAs (lncRNAs) and micro RNAs (miRNAs). Our analysis revealed a total of 170, 118, 99, and 269 differentially expressed lncRNA and 120, 118, 99, and 124 differentially expressed miRNAs in 2, 4, 6 and 8 ppt treatment groups respectively. Two competing endogenous RNA (ceRNA) networks were constructed i.e. A* ceRNA network with up-regulated lncRNAs and mRNAs, down-regulated miRNAs; and B* ceRNA network vice versa. 2ppt group had 131 and 83 lncRNA-miRNA-mRNA pairs in A* and B* networks, respectively. 4ppt group featured 163 pairs in A* network and 191 in B* network, while the 6ppt had 103 and 105 pairs. 8ppt group included 192 and 174 pairs. These networks illuminate the intricate RNA interactions in freshwater fish to varying salinity conditions.

Decryption of sequence, structure, and functional features of SINE repeat elements in SINEUP non-coding RNA-mediated post-transcriptional gene regulation

RNA structure folding largely influences RNA regulation by providing flexibility and functional diversity. In silico and in vitro analyses are limited in their ability to capture the intricate relationships between dynamic RNA structure and RNA functional diversity present in the cell. Here, we investigate sequence, structure and functional features of mouse and human SINE-transcribed retrotransposons embedded in SINEUPs long non-coding RNAs, which positively regulate target gene expression post-transcriptionally. In-cell secondary structure probing reveals that functional SINEs-derived RNAs contain conserved short structure motifs essential for SINEUP-induced translation enhancement. We show that SINE RNA structure dynamically changes between the nucleus and cytoplasm and is associated with compartment-specific binding to RBP and related functions. Moreover, RNA–RNA interaction analysis shows that the SINE-derived RNAs interact directly with ribosomal RNAs, suggesting a mechanism of translation regulation. We further predict the architecture of 18 SINE RNAs in three dimensions guided by experimental secondary structure data. Overall, we demonstrate that the conservation of short key features involved in interactions with RBPs and ribosomal RNA drives the convergent function of evolutionarily distant SINE-transcribed RNAs.

Global identification of mRNA-interacting circular RNAs by CLiPPR-Seq.

Although the functional role of circular RNA (circRNA) interaction with microRNAs and proteins has been studied extensively, circRNA interactions with the protein-coding mRNAs in intact cells remain largely unknown. Here, by employing AMT-mediated proximity ligation of RNA-RNA duplexes followed by circRNA enrichment and deep sequencing, we report a novel Cross-Linking Poly(A) Pulldown RNase R Sequencing (CLiPPR-seq) technology which identified hundreds of mRNA-interacting circRNAs in three different cell types, including βTC6, C2C12and HeLa cells. Furthermore, CLiPP-seq without RNase R treatment was also performed to identify the mRNA expression in these cells. BLAST analysis of circRNAs in CLiPPR-seq samplewith the mRNAs in CLiPP-seq samples determined their potential complementary sequences for circRNA-mRNA interaction. Pulldown of circRNAs and poly(A) RNAs confirmed the direct interaction of circRNAs with target mRNAs. Silencing of mRNA-interacting circRNAs led to the altered expression of target mRNAs in βTC6 cells, suggesting the role of direct interaction of circRNAs with mRNAs in gene expression regulation. CLiPPR-seq thus represents a novel method for illuminating the myriad of uncharacterized circRNA-mRNA hybrids that may regulate gene expression.