Critical role of backbone coordination in the mRNA recognition by RNA induced silencing complex

Despite its functional importance, the molecular mechanism underlying target mRNA recognition by Argonaute (Ago) remains largely elusive. Based on extensive all-atom molecular dynamics simulations, we constructed quasi-Markov State Model (qMSM) to reveal the dynamics during recognition at position 6-7 in the seed region of human Argonaute 2 (hAgo2). Interestingly, we found that the slowest mode of motion therein is not the gRNA-target base-pairing, but the coordination of the target phosphate groups with a set of positively charged residues of hAgo2. Moreover, the ability of Helix-7 to approach the PIWI and MID domains was found to reduce the effective volume accessible to the target mRNA and therefore facilitate both the backbone coordination and base-pair formation. Further mutant simulations revealed that alanine mutation of the D358 residue on Helix-7 enhanced a trap state to slow down the loading of target mRNA. Similar trap state was also observed when wobble pairs were introduced in g6 and g7, indicating the role of Helix-7 in suppressing non-canonical base-paring. Our study pointed to a general mechanism for mRNA recognition by eukaryotic Agos and demonstrated the promise of qMSM in investigating complex conformational changes of biomolecular systems.

Binding induced functional domain motions in the argonaute characterized by adaptive advanced sampling

Argonaute proteins in combination with short microRNA (miRNAs) can target mRNA molecules for translation inhibition or degradation and play a key role in many regulatory processes. The miRNAs act as guide RNAs that associate with Argonaute and the complementary mRNA target region. The complex formation results in activation of Argonaute and specific cleavage of the target mRNA. Both the binding and activation processes involve essential domain rearrangements of functional importance. For the Thermus Thermophilus Argonaute (TtAgo) system guide-bound (binary) and guide/target-bound (ternary) complexes are known but how the binding of guide and target mediate domain movements is still not understood. We have studied the Argonaute domain motion in apo and guide/target bound states using Molecular Dynamics simulations and a Hamiltonian replica exchange (H-REMD) method that employs a specific biasing potential to accelerate domain motions. The H-REMD technique indicates sampling of a much broader distribution of domain arrangements both in the apo as well as binary and ternary complexes compared to regular MD simulations. In the apo state domain arrangements corresponding to more compact (closed) states are mainly sampled which undergo an opening upon guide and guide/target binding. Whereas only limited overlap in domain geometry between apo and bound states was found, a larger similarity in the domain distribution is observed for the simulations of binary and ternary complexes. Comparative simulations on ternary complexes with 15 or 16 base pairs (bp) formed between guide and target strands (instead of 14) resulted in dissociation of the 3’-guide strand from the PAZ domain and domain rearrangement. This agrees with the experimental observation that guide-target pairing beyond 14 bps is required for activation and gives a mechanistic explanation for the experimentally observed activation process.

The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation

Gene regulation via N6-methyladenosine (m6A) in mRNA involves RNA-binding proteins that recognize m6A via a YT521-B homology (YTH) domain. The plant YTH domain proteins ECT2 and ECT3 act genetically redundantly in stimulating cell proliferation during organogenesis, but several fundamental questions regarding their mode of action remain unclear. Here, we use HyperTRIBE (targets of RNA-binding proteins identified by editing) to show that most ECT2 and ECT3 targets overlap, with only few examples of preferential targeting by either of the two proteins. HyperTRIBE in different mutant backgrounds also provides direct views of redundant and specific target interactions of the two proteins. We also show that contrary to conclusions of previous reports, ECT2 does not accumulate in the nucleus. Accordingly, inactivation of ECT2, ECT3 and their surrogate ECT4 does not change patterns of polyadenylation site choice in ECT2/3 target mRNAs, but does lead to lower steady state accumulation of target mRNAs. In addition, mRNA and microRNA expression profiles show indications of stress response activation in ect2/ect3/ect4 mutants, likely via indirect effects. Thus, previous suggestions of control of alternative polyadenylation by ECT2 are not supported by evidence, and ECT2 and ECT3 act largely redundantly to regulate target mRNA, including its abundance, in the cytoplasm.

Comprehensive Analysis of Potential miRNA-Target mRNA-Immunocyte Subtype Network in Cerebral Infarction

<b><i>Introduction:</i></b> Cerebral infarction (CI) is one of the leading causes of serious long-term disability and mortality. <b><i>Objective:</i></b> We aimed to identify potential miRNAs and target mRNAs and assess the involvement of immunocyte infiltration in the process of CI. <b><i>Methods:</i></b> First, miRNA and mRNA data were downloaded from the Gene Expression Omnibus database, followed by differential expression analysis. Second, correlation analysis between differentially expressed mRNAs and differential immunocyte subtypes was performed through the CIBERSORT algorithm. Third, the regulatory network between miRNAs and immunocyte subtype-related mRNAs was constructed followed by the functional analysis of these target mRNAs. Fourth, correlation validation between differentially expressed mRNAs and differential immunocyte subtypes was performed in the GSE37587 dataset. Finally, the diagnostic ability of immunocyte subtype-related mRNAs was tested. <b><i>Results:</i></b> Up to 17 differentially expressed miRNAs and 3,267 differentially expressed mRNAs were identified, among which 310 differentially expressed mRNAs were significantly associated with immunocyte subtypes. Several miRNA-target mRNA-immunocyte subtype networks including hsa-miR-671-3p-ZC3HC1-neutrophils, hsa-miR-625-CD5-monocytes, hsa-miR-122-ACOX1/DUSP1/NEDD9-neutrophils, hsa-miR-455-5p-SLC24A4-monocytes, and hsa-miR-455-5p-SORL1-neutrophils were identified. LAT, ACOX1, DUSP1, NEDD9, ZC3HC1, BIN1, AKT1, DNMT1, SLC24A4, and SORL1 had a potential diagnostic value for CI. <b><i>Conclusions:</i></b> The network including miRNA, target mRNA, and immunocyte subtype may be novel regulators and diagnostic and therapeutic targets in CI.

The Arabidopsis m6A-binding proteins ECT2 and ECT3 bind largely overlapping mRNA target sets and influence target mRNA abundance, not alternative polyadenylation

Gene regulation via N6-methyladenosine (m6A) in mRNA involves RNA-binding proteins that recognize m6A via a YT521-B homology (YTH) domain. The plant YTH domain proteins ECT2 and ECT3 act genetically redundantly in stimulating cell proliferation during organogenesis, but several fundamental questions regarding their mode of action remain unclear. Here, we use HyperTRIBE (targets of RNA-binding proteins identified by editing) to show that most ECT2 and ECT3 targets overlap, with only few examples of preferential targeting by either of the two proteins. HyperTRIBE in different mutant backgrounds also provides direct views of redundant and specific target interactions of the two proteins. We also show that contrary to conclusions of previous reports, ECT2 does not accumulate in the nucleus. Accordingly, inactivation of ECT2, ECT3 and their surrogate ECT4 does not change patterns of polyadenylation site choice in ECT2/3 target mRNAs, but does lead to lower steady state accumulation of target mRNAs. In addition, mRNA and microRNA expression profiles show indications of stress response activation in ect2/ect3/ect4 mutants, likely via indirect effects. Thus, previous suggestions of control of alternative polyadenylation by ECT2 are not supported by evidence, and ECT2 and ECT3 act largely redundantly to regulate target mRNA, including its abundance, in the cytoplasm.

The Cell Fate Determinant Musashi Is Controlled Through Dynamic Protein:Protein Interactions

The Musashi RNA-binding protein functions as a gatekeeper of cell maturation and plasticity through the control of target mRNA translation. It is understood that Musashi promotes stem cell self-renewal and opposes differentiation. While Musashi is best characterized as a repressor of target mRNA translation, we have shown that Musashi can activate target mRNA translation in a cell context specific manner via regulatory phosphorylation on two evolutionarily conserved C-terminal serine residues. Our recent work has found that Musashi is expressed in pituitary stem cells as well as in differentiated hormone producing cell lineages in the adult pituitary. We hypothesize that Musashi maintains cell fate plasticity in the adult pituitary to allow the gland to modulate hormone production in response to changing organismal needs. Here, we seek to understand the regulation of Musashi function. Both Musashi isoforms (Musashi1 and Musashi2) contain two RNA-recognition motifs (RRMs) that bind to specific sequences in the 3’-UTR of target mRNA transcripts; however, neither isoform has enzymatic properties and thus functions through interactions with other proteins to regulate translational outcomes, but the identity and role of Musashi partner proteins is largely unknown. In this study, we have identified co-associated partner proteins that functionally contribute to Musashi-dependent mRNA translational activation during the maturation of Xenopus oocytes. Using mass spectrometry, we identified 29 co-associated proteins that interact specifically with Musashi1 during oocyte maturation and determined that the Musashi co-associated proteins ePABP, PABP4, LSM14A/B, CELF2, PUM1, ELAV1, ELAV2, and DDX6 attenuated oocyte maturation through individual antisense DNA oligo knockdowns. An assessment of the role of these cofactors in the control of Musashi-dependent target mRNA translation is in progress. In addition to studying co-associated proteins, we have created a computational 3D model of the Musashi1 molecule to assist in our investigation Musashi dimerization. This model has indicated that both Musashi1 dimerization and Musashi1:Musashi2 heterodimerization are energetically favorable, and co-pulldown studies have verified both Musashi1 homo-dimerization and Musashi1:Musashi2 heterodimerization in vivo. Computational modeling of Musashi dimer complexes has also identified the key amino acids necessary for these interactions. The contribution of each co-associated protein’s influence on Musashi-dependent translation, relative to the requirement for Musashi:Musashi dimerization, is expected to provide unparalleled insight into regulation of Musashi action. Moreover, cell type specific regulation of association of Musashi co-factors would directly influence Musashi target mRNA translation in oocyte maturation and during pituitary cell plasticity.

The RNA-binding protein RBP42 regulates cellular energy metabolism in mammalian-infective Trypanosoma brucei

RNA-binding proteins are key players in coordinated post-transcriptional regulation of functionally related genes, defined as RNA regulons. RNA regulons play particularly critical roles in parasitic trypanosomes, which exhibit unregulated co-transcription of long arrays of unrelated genes. In this report, we present a systematic analysis of an essential RNA-binding protein, RBP42, in the mammalian-infective slender bloodstream form of African trypanosome, and we show that RBP42 is a key regulator of parasite’s central carbon and energy metabolism. Using individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to identify genome-wide RBP42-RNA interactions, we show that RBP42 preferentially binds within the coding region of mRNAs encoding core metabolic enzymes. Using global quantitative transcriptomic and proteomic analyses, we also show that loss of RBP42 reduces the abundance of target mRNA-encoded proteins, but not target mRNA, suggesting a plausible role of RBP42 as a positive regulator of target mRNA translation. Analysis reveals significant changes in central carbon metabolic intermediates following loss of RBP42, further supporting its critical role in cellular energy metabolism.

MicroRNA-4429 suppresses proliferation of prostate cancer cells by targeting distal-less homeobox 1 and inactivating the Wnt/β-catenin pathway

Background Emerging evidence suggests that microRNAs (miRNAs) play multiple roles in human cancers through regulating mRNAs and distinct pathways. This paper focused on the functions of miR-4429 in prostate cancer (PCa) progression and the molecules involved. Methods Expression of miR-4429 in PCa tissues and cells was determined. Upregulation of miR-4429 was introduced in PCa cells to examine its role in the malignant behaviors of cells. The putative target mRNA of miR-4429 involved in PCa progression was predicted from a bioinformatic system and validated through luciferase assays. Overexpression of distal-less homeobox 1 (DLX1) was further induced in cells to validate its implication in miR-4429-mediated events. The activity of Wnt/β-catenin pathway was determined. Results miR-4429 was poorly expressed in PCa tissues and cells. Artificial upregulation of miR-4429 significantly reduced proliferation, growth, invasion, migration and resistance to death of cancer cells and inactivated the Wnt/β-catenin pathway. DLX1 mRNA was found as a target of miR-4429. Upregulation of DLX1 restored the malignant behaviors of PCa cells which were initially suppressed by miR-4429, and it activated the Wnt/β-catenin pathway. Conclusion Our study highlights that miR-4429 inhibits the growth of PCa cells by down-regulating DLX1 and inactivating the Wnt/β-catenin pathway. This finding may offer novel insights into PCa treatment.

The Emerging Impact of RNA Editing in Tumor Growth and Metastasis

Context: RNA editing is an essential modification that needs to develop normal cells and is involved in a wide range of biological processes. It can arise in both coding and non-coding sequences with different functional effects. Although the expansion of transcriptome diversity is the primary goal of RNA editing, dysregulation and aberrant editing may act as an essential contributor to cancer pathogenesis. Evidence Acquisition: The current review aimed to investigate the role of RNA editing in cancer initiation and progression. Science Direct and PubMed databases were reviewed from 2000 to 2020 and 2003 to 2020, respectively, using various combinations of "RNA editing" and "cancer" keywords. Results: The location of editing sites has different functional impacts on tumorigenesis. Nonsynonymous editing in antizyme inhibitor 1 (AZIN1) leads to a metastatic progression of colorectal and gastric cancer. Recoding editing events in bladder cancer-associated protein (BLCAP) is correlated with the progression of cervical carcinogenesis. Editing events located at 3′UTRs are also a general mechanism to promote tumor growth in different types of cancers. A significant number of editing events in microRNAs with a functional role in cancer are also reported. These editing sites could change the fate and function of microRNAs, either by preventing target mRNA recognition or by dysregulating an off-target mRNA. Conclusions: There are increasing shreds of evidence on the key role of RNA editing events in cancer initiation and progression.

Effect of miR-21 on Renal Fibrosis Induced by Nano-SiO2 in Diabetic Nephropathy Rats via PTEN/AKT Pathway

MicroRNAs are a type of non-coding single-stranded RNA that can mediate target mRNA degradation or inhibit target mRNA translation, thereby regulating target gene expression and have an important role in physiological and pathological processes. At present, miRs have been confirmed to be closely related to kidneys and kidney diseases, and have been involved in the occurrence, development and prognosis of renal fibrosis. Now we review the research progress of miRs in renal fibrosis in recent years, and provide references for the future diagnosis and treatment of renal fibrosis. The incidence of diabetic nephropathy (DN) is increasing year by year, the pathogenesis is complicated, and renal fibrosis occurs during the progress of the disease, which is very difficult to treat. The protein encoded by the PTEN gene has lipid phosphatase and protein phosphatase activity and is the PTEN/AKT and FAK pathway important negative regulators. It can play an anti-fibrotic effect by negatively regulating the PTEN/AKT pathway. Studies show that during the pathogenesis of DN, the expression of PTEN protein is reduced, and the PI3K/AKT pathway is activated to exert multiple fibrotic effects, but affect PTEN. The regulatory factors of expression are still not clear; moreover, the specific mechanism of the decrease in PTEN protein expression in DN pathogenesis. Therefore, this study intends to Intervention of the expression level of miRs in renal tissues, to study its regulation of PTEN and its effect on renal fibrosis, and at the same time, observe the effects on renal tubular epithelial cell phenotype and fibrotic lesions under high glucose conditions by up-regulating and down-regulating PTEN expression. Further elucidate the pathogenesis of DN renal fibrosis, and explore new effective targets for the prevention and treatment of DN.