Stabilization of RRBP1 mRNA via an m6A-dependent manner in prostate cancer constitutes a therapeutic vulnerability amenable to small-peptide inhibition of METTL3.

Mounting evidence has implicated the RNA m6A methylation catalyzed by METTL3 in a wide range of physiological and pathological processes, including tumorigenesis. The detailed m6A landscape and molecular mechanism of METTL3 in prostate cancer (PCa) remains ill-defined. We find that METTL3 is overexpressed in PCa and correlates with worse patient survival. Functional studies establish METTL3 as an oncoprotein dependent on its m6A enzymatic activity in both AR+ and AR- PCa cells. To dissect the regulatory network of m6A pathway in PCa, we map the m6A landscape in clinical tumor samples using m6A-seq and identify genome-wide METTL3-binding transcripts via RIP-seq. Mechanistically, we discover RRBP1 as a direct METTL3 target in which METTL3 stabilizes RRBP1 mRNA in an m6A-dependent manner. RRBP1 positively correlates with METTL3 expression in PCa cohorts and exerts an oncogenic role in aggressive PCa cells. Leveraging the 3D structural protein-protein interaction between METTL3 and METTL14, we successfully develop two potential METTL3 peptide inhibitors (RM3 and RSM3) that effectively suppress cancer cell proliferation in vitro and tumor growth in vivo. Collectively, our study reveals a novel METTL3/m6A/RRBP1 axis in enhancing aggressive traits of PCa, which can be therapeutically targeted by small-peptide METTL3 antagonists.

Non-redundant roles for the human mRNA decapping cofactor paralogs DCP1a and DCP1b.

Eukaryotic gene expression is regulated at the transcriptional and post-transcriptional levels, with disruption of regulation contributing significantly to human diseases. The 5' m7G mRNA cap is a central node in post-transcriptional regulation, participating in both mRNA stabilization and translation efficiency. In mammals, DCP1a and DCP1b are paralogous cofactor proteins of the mRNA cap hydrolase DCP2. As lower eukaryotes have a single DCP1 cofactor, the functional advantages gained by this evolutionary divergence remain unclear. We report the first functional dissection of DCP1a and DCP1b, demonstrating that they are non-redundant cofactors of DCP2 with unique roles in decapping complex integrity and specificity. DCP1a is essential for decapping complex assembly and interactions between the decapping complex and mRNA cap-binding proteins. DCP1b is essential for decapping complex interactions with protein degradation and translational machinery. DCP1a and DCP1b impact the turnover of distinct mRNAs. The observation that different ontological groups of mRNA molecules are regulated by DCP1a and DCP1b, along with their non-redundant roles in decapping complex integrity, provides the first evidence that these paralogs have qualitatively distinct functions.

The SIX2/PFN2 feedback loop promotes the stemness of gastric cancer cells.

BACKGROUND: The roles of the transcriptional factor SIX2 have been identified in several tumors. However, its roles in gastric cancer (GC) progression have not yet been revealed. Our objective is to explore the impact and underlying mechanisms of SIX2 on the stemness of GC cells. METHODS: Lentivirus infection was employed to establish stable expression SIX2 or PFN2 in GC cells. Gain- and loss-of-function experiments were conducted to detect changes of stemness markers, flow cytometry profiles, tumor spheroid formation, and tumor-initiating ability. ChIP, RNA-sequencing, tissue microarray, and bioinformatics analysis were performed to reveal the correlation between SIX2 and PFN2. The mechanisms underlying the SIX2/PFN2 loop-mediated effects were elucidated through tissue microarray analysis, RNA stability assay, IP-MS, Co-Immunoprecipitation, and inhibition of the JNK signaling pathway. RESULTS: The stemness of GC cells was enhanced by SIX2. Mechanistically, SIX2 directly bound to PFN2's promoter and promoted PFN2 activity. PFN2, in turn, promoted the mRNA stability of SIX2 by recruiting RNA binding protein YBX-1, subsequently activating the downstream MAPK/JNK pathway. CONCLUSION: This study unveils the roles of SIX2 in governing GC cell stemness, defining a novel SIX2/PFN2 regulatory loop responsible for this regulation. This suggests the potential of targeting the SIX2/PFN2 loop for GC treatment (Graphical Abstracts).

Genetic polymorphism in untranslated regions of PRKCZ influences mRNA structure, stability and binding sites.

BACKGROUND: Variations in untranslated regions (UTR) alter regulatory pathways impacting phenotype, disease onset, and course of disease. Protein kinase C Zeta (PRKCZ), a serine-threonine kinase, is implicated in cardiovascular, neurological and oncological disorders. Due to limited research on PRKCZ, this study aimed to investigate the impact of UTR genetic variants' on binding sites for transcription factors and miRNA. RNA secondary structure, eQTLs, and variation tolerance analysis were also part of the study. METHODS: The data related to PRKCZ gene variants was downloaded from the Ensembl genome browser, COSMIC and gnomAD. The RegulomeDB database was used to assess the functional impact of 5' UTR and 3'UTR variants. The analysis of the transcription binding sites (TFBS) was done through the Alibaba tool, and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) was employed to identify pathways associated with PRKCZ. To predict the effect of variants on microRNA binding sites, PolymiRTS was utilized for 3' UTR variants, and the SNPinfo tool was used for 5' UTR variants. RESULTS: The results obtained indicated that a total of 24 variants present in the 3' UTR and 25 variants present in the 5' UTR were most detrimental. TFBS analysis revealed that 5' UTR variants added YY1, repressor, and Oct1, whereas 3' UTR variants added AP-2alpha, AhR, Da, GR, and USF binding sites. The study predicted TFs that influenced PRKCZ expression. RNA secondary structure analysis showed that eight 5' UTR and six 3' UTR altered the RNA structure by either removal or addition of the stem-loop. The microRNA binding site analysis highlighted that seven 3' UTR and one 5' UTR variant altered the conserved site and also created new binding sites. eQTLs analysis showed that one variant was associated with PRKCZ expression in the lung and thyroid. The variation tolerance analysis revealed that PRKCZ was an intolerant gene. CONCLUSION: This study laid the groundwork for future studies aimed at targeting PRKCZ as a therapeutic target.

Pleiotropic effects of PAB1 deletion: Extensive changes in the yeast proteome, transcriptome, and translatome.

Cytoplasmic poly(A)-binding protein (PABPC; Pab1 in yeast) is thought to be involved in multiple steps of post-transcriptional control, including translation initiation, translation termination, and mRNA decay. To understand both the direct and indirect roles of PABPC in more detail, we have employed mass spectrometry to assess the abundance of the components of the yeast proteome, as well as RNA-Seq and Ribo-Seq to analyze changes in the abundance and translation of the yeast transcriptome, in cells lacking the PAB1 gene. We find that pab1Δ cells manifest drastic changes in the proteome and transcriptome, as well as defects in translation initiation and termination. Defects in translation initiation and the stabilization of specific classes of mRNAs in pab1Δ cells appear to be partly indirect consequences of reduced levels of specific initiation factors, decapping activators, and components of the deadenylation complex in addition to the general loss of Pab1's direct role in these processes. Cells devoid of Pab1 also manifested a nonsense codon readthrough phenotype indicative of a defect in translation termination. Collectively, our results indicate that, unlike the loss of simpler regulatory proteins, elimination of cellular Pab1 is profoundly pleiotropic and disruptive to numerous aspects of post-transcriptional regulation.

Critical factors for precise and efficient RNA cleavage by RNase Y in Staphylococcus aureus.

Cellular processes require precise and specific gene regulation, in which continuous mRNA degradation is a major element. The mRNA degradation mechanisms should be able to degrade a wide range of different RNA substrates with high efficiency, but should at the same time be limited, to avoid killing the cell by elimination of all cellular RNA. RNase Y is a major endoribonuclease found in most Firmicutes, including Bacillus subtilis and Staphylococcus aureus. However, the molecular interactions that direct RNase Y to cleave the correct RNA molecules at the correct position remain unknown. In this work we have identified transcripts that are homologs in S. aureus and B. subtilis, and are RNase Y targets in both bacteria. Two such transcript pairs were used as models to show a functional overlap between the S. aureus and the B. subtilis RNase Y, which highlighted the importance of the nucleotide sequence of the RNA molecule itself in the RNase Y targeting process. Cleavage efficiency is driven by the primary nucleotide sequence immediately downstream of the cleavage site and base-pairing in a secondary structure a few nucleotides downstream. Cleavage positioning is roughly localised by the downstream secondary structure and fine-tuned by the nucleotide immediately upstream of the cleavage. The identified elements were sufficient for RNase Y-dependent cleavage, since the sequence elements from one of the model transcripts were able to convert an exogenous non-target transcript into a target for RNase Y.

What goes up must come down: off switches for regulatory RNAs.

Small RNAs base pair with and regulate mRNA translation and stability. For both bacterial small regulatory RNAs and eukaryotic microRNAs, association with partner proteins is critical for the stability and function of the regulatory RNAs. We review the mechanisms for degradation of these RNAs: displacement of the regulatory RNA from its protein partner (in bacteria) or destruction of the protein and its associated microRNAs (in eukaryotes). These mechanisms can allow specific destruction of a regulatory RNA via pairing with a decay trigger RNA or function as global off switches by disrupting the stability or function of the protein partner.

ATF1 promotes ferroptosis resistance in lung cancer through enhancing mRNA stability of PROM2.

BACKGROUND: Ferroptotic proteins are promising therapeutic targets for lung cancer. The PROM2 is upregulated in lung cancer and known to suppress ferroptosis. This study examined the molecular mechanisms for PROM2-induced ferroptosis resistance in lung cancer. METHODS: Ferroptosis in lung cancer was assessed by iron kit, and transmission electron microscopy was applied to observe the changes in mitochondrial morphology. BODIPY™ was applied to test the lipid ROS, and MeRIP was performed to test the m6A modification of PROM2. RIP assay was employed for confirming the binding between METTL3 and PROM2. In addition, dual luciferase assay was employed for exploring the transcriptional regulation of ATF1 to METTL3, and the binding relation between ATF1 and METTL3 promoter region was explored by ChIP assay. RESULTS: Expression levels of PROM2 were significantly higher in lung cancer cell lines than a noncancerous control line, and PROM2 knockdown significantly reduced both cancer cell viability and proliferation rate. In addition, PROM2 knockdown reduced xenograft tumor growth and exacerbated erastin-induced ferroptosis. Compared to PROM2 mRNA from control cells, transcripts in lung cancer cells exhibited enhanced m6A levels, and showed greater binding with METTL3. Further, ATF1 upregulated METTL3 transcription, thereby stabilizing PROM2 mRNA and increasing ferroptosis resistance. CONCLUSION: ATF1 could promote ferroptosis resistance in lung cancer through enhancing mRNA stability of PROM2. Thus, our work might shed novel insights on discovering therapeutic strategy for lung cancer.

METTL5 enhances the mRNA stability of TPRKB through m6A modification to facilitate the aggressive phenotypes of hepatocellular carcinoma cell.

N6-methyladenosine (m6A) modification plays an important role in RNA molecular functions, therefore affecting the initiation and development of hepatocellular carcinoma (HCC). Herein, multiple datasets were applied to conduct a comprehensive analysis of DEGs within HCC and the analysis revealed significant dysregulation of numerous genes. Functional and signaling pathway enrichment analyses were performed. Further, TP53RK binding protein (TPRKB) emerged as a significant factor, exhibiting high expression level within HCC tissue samples and cells which could predict HCC patients' poor OS. Knockdown investigations of TPRKB in vitro demonstrated the effect of TPRKB knockdown on attenuating the aggressiveness of HCC cells by suppressing the viability, colony formation, invasive ability, and migratory ability, inducing cell cycle arrest, and facilitating the apoptosis of HCC cells. Investigations in vivo revealed that TPRKB knockdown significantly suppressed tumor growth in mice model. Additionally, the study identified methyltransferase 5, N6-adenosine (METTL5) as a potential regulator of TPRKB expression via m6A modification, positively regulating TPRKB expression by enhancing TPRKB mRNA stability. The dynamic effects of METTL5 and TPRKB upon the phenotypes of HCC cells further confirmed that TPRKB overexpression partially abolished the anti-cancer effects of METTL5 knockdown upon the aggressiveness of HCC cells. Conclusively, our findings uncover that TPRKB, significantly overexpressed in HCC, exerts a critical effect on promoting tumor aggressiveness, and its expression shows to be positively regulated by METTL5 via m6A methylation. These insights deepen the understanding of HCC pathogenesis and open new avenues for targeted therapies, highlighting that METTL5-TPRKB axis is an underlying new therapeutic target in HCC management.

Comparative analyses suggest a link between mRNA splicing, stability, and RNA covalent modifications in flowering plants.

BACKGROUND: In recent years, covalent modifications on RNA nucleotides have emerged as pivotal moieties influencing the structure, function, and regulatory processes of RNA Polymerase II transcripts such as mRNAs and lncRNAs. However, our understanding of their biological roles and whether these roles are conserved across eukaryotes remains limited. RESULTS: In this study, we leveraged standard polyadenylation-enriched RNA-sequencing data to identify and characterize RNA modifications that introduce base-pairing errors into cDNA reads. Our investigation incorporated data from three Poaceae (Zea mays, Sorghum bicolor, and Setaria italica), as well as publicly available data from a range of stress and genetic contexts in Sorghum and Arabidopsis thaliana. We uncovered a strong enrichment of RNA covalent modifications (RCMs) deposited on a conserved core set of nuclear mRNAs involved in photosynthesis and translation across these species. However, the cohort of modified transcripts changed based on environmental context and developmental program, a pattern that was also conserved across flowering plants. We determined that RCMs can partly explain accession-level differences in drought tolerance in Sorghum, with stress-associated genes receiving a higher level of RCMs in a drought tolerant accession. To address function, we determined that RCMs are significantly enriched near exon junctions within coding regions, suggesting an association with splicing. Intriguingly, we found that these base-pair disrupting RCMs are associated with stable mRNAs, are highly correlated with protein abundance, and thus likely associated with facilitating translation. CONCLUSIONS: Our data point to a conserved role for RCMs in mRNA stability and translation across the flowering plant lineage.

The m6A reader IGF2BP2 promotes ESCC progression by stabilizing HDGF mRNA.

OBJECTIVE: This study aimed to explore the role of IGF2BP2 in esophageal squamous cell carcinoma (ESCC) progression. MATERIALS AND METHODS: The Cancer Genome Atlas (TCGA) dataset, transcriptome sequencing, and the Gene Expression Omnibus (GEO) dataset were used to detect the expression of m6A-associated genes in ESCC. The in vitro and in vivo assays were used to explore the role of IGF2BP2 in ESCC. RESULTS: IGF2BP2 was significantly overexpressed in human ESCC specimens, which was confirmed by analyzing the GEO dataset. IGF2BP2 overexpression was correlated with poor prognosis in patients with ESCC. Altering the expression of IGF2BP2 influenced the proliferation, migration, and invasion of ESCC cells in vitro and tumorigenicity in vivo. IGF2BP2 could bind to and stabilize hepatoma-derived growth factor (HDGF) transcripts in ESCC in an m6A-dependent manner and promote HDGF expression. CONCLUSIONS: These findings indicate that the novel IGF2BP2-HDGF axis is pivotal for ESCC cancer progression and can serve as a target for developing therapeutics.

ILP1 and NTR1 affect the stability of U6 snRNA during spliceosome complex disassembly in Arabidopsis.

U6 snRNA is one of the uridine-rich non-coding RNAs, abundant and stable in various cells, function as core particles in the intron-lariat spliceosome (ILS) complex. The Increased Level of Polyploidy1-1D (ILP1) and NTC-related protein 1 (NTR1), two conserved disassembly factors of the ILS complex, facilitates the disintegration of the ILS complex after completing intron splicing. The functional impairment of ILP1 and NTR1 lead to increased U6 levels, while other snRNAs comprising the ILS complex remained unaffected. We revealed that ILP1 and NTR1 had no impact on the transcription, 3' end phosphate structure or oligo(U) tail of U6 snRNA. Moreover, we uncovered that the mutation of ILP1 and NTR1 resulted in the accumulation of ILS complexes, impeding the dissociation of U6 from splicing factors, leading to an extended half-life of U6 and ultimately causing an elevation in U6 snRNA levels. Our findings broaden the understanding of the functions of ILS disassembly factors ILP1 and NTR1, and providing insights into the dynamic disassembly between U6 and ILS.

DIS3 ribonuclease is essential for spermatogenesis and male fertility in mice.

Spermatogonial stem cell (SSC) self-renewal and differentiation provide foundational support for long-term, steady-state spermatogenesis in mammals. Here, we have investigated the essential role of RNA exosome associated DIS3 ribonuclease in maintaining spermatogonial homeostasis and facilitating germ cell differentiation. We have established male germ-cell Dis3 conditional knockout (cKO) mice in which the first and subsequent waves of spermatogenesis are disrupted. This leads to a Sertoli cell-only phenotype and sterility in adult male mice. Bulk RNA-seq documents that Dis3 deficiency partially abolishes RNA degradation and causes significant increases in the abundance of transcripts. This also includes pervasively transcribed PROMoter uPstream Transcripts (PROMPTs), which accumulate robustly in Dis3 cKO testes. In addition, scRNA-seq analysis indicates that Dis3 deficiency in spermatogonia significantly disrupts RNA metabolism and gene expression, and impairs early germline cell development. Overall, we document that exosome-associated DIS3 ribonuclease plays crucial roles in maintaining early male germ cell lineage in mice.

Staphylococcal exoribonuclease YhaM destabilizes ribosomes by targeting the mRNA of a hibernation factor.

The hibernation-promoting factor (Hpf) in Staphylococcus aureus binds to 70S ribosomes and induces the formation of the 100S complex (70S dimer), leading to translational avoidance and occlusion of ribosomes from RNase R-mediated degradation. Here, we show that the 3'-5' exoribonuclease YhaM plays a previously unrecognized role in modulating ribosome stability. Unlike RNase R, which directly degrades the 16S rRNA of ribosomes in S. aureus cells lacking Hpf, YhaM destabilizes ribosomes by indirectly degrading the 3'-hpf mRNA that carries an intrinsic terminator. YhaM adopts an active hexameric assembly and robustly cleaves ssRNA in a manganese-dependent manner. In vivo, YhaM appears to be a low-processive enzyme, trimming the hpf mRNA by only 1 nucleotide. Deletion of yhaM delays cell growth. These findings substantiate the physiological significance of this cryptic enzyme and the protective role of Hpf in ribosome integrity, providing a mechanistic understanding of bacterial ribosome turnover.

Enhancers associated with unstable RNAs are rare in plants.

Unstable transcripts have emerged as markers of active enhancers in vertebrates and shown to be involved in many cellular processes and medical disorders. However, their prevalence and role in plants is largely unexplored. Here, we comprehensively captured all actively initiating (nascent) transcripts across diverse crops and other plants using capped small (cs)RNA sequencing. We discovered that unstable transcripts are rare in plants, unlike in vertebrates, and when present, often originate from promoters. In addition, many 'distal' elements in plants initiate tissue-specific stable transcripts and are likely bona fide promoters of as-yet-unannotated genes or non-coding RNAs, cautioning against using reference genome annotations to infer putative enhancer sites. To investigate enhancer function, we integrated data from self-transcribing active regulatory region (STARR) sequencing. We found that annotated promoters and other regions that initiate stable transcripts, but not those marked by unstable or bidirectional unstable transcripts, showed stronger enhancer activity in this assay. Our findings underscore the blurred line between promoters and enhancers and suggest that cis-regulatory elements can encompass diverse structures and mechanisms in eukaryotes, including humans.

NOP2 facilitates EZH2-mediated epithelial-mesenchymal transition by enhancing EZH2 mRNA stability via m5C methylation in lung cancer progression.

NOP2, a member of the NOL1/NOP2/SUN domain (NSUN) family, is responsible for catalyzing the posttranscriptional modification of RNA through 5-methylcytosine (m5C). Dysregulation of m5C modification has been linked to the pathogenesis of various malignant tumors. Herein, we investigated the expression of NOP2 in lung adenocarcinoma (LUAD) tissues and cells, and found that it was significantly upregulated. Moreover, lentivirus-mediated overexpression of NOP2 in vitro resulted in enhanced migration and invasion capabilities of lung cancer cells, while in vivo experiments demonstrated its ability to promote the growth and metastasis of xenograft tumors. In contrast, knockdown of NOP2 effectively inhibited the growth and metastasis of lung cancer cells. RNA-sequencing was conducted to ascertain the downstream targets of NOP2, and the findings revealed a significant upregulation in EZH2 mRNA expression upon overexpression of NOP2. Subsequent validation experiments demonstrated that NOP2 exerted an m5C-dependent influence on the stability of EZH2 mRNA. Additionally, our investigations revealed a co-regulatory relationship between NOP2 and the m5C reader protein ALYREF in modulating the stability of EZH2 mRNA. Notably, the NOP2/EZH2 axis facilitated the malignant phenotype of lung cancer cells by inducing epithelial-mesenchymal transition (EMT) both in vitro and in vivo. Mechanistically, ChIP analysis proved that EZH2 counteracted the impact of NOP2 on the occupancy capacity of EZH2 and H3K27me3 in the promoter regions of E-cadherin, a gene crucial for regulating EMT. In a word, our research highlights the significant role of NOP2 in LUAD and offers novel mechanistic insights into the NOP2/ALYREF/EZH2 axis, which holds promise as a potential target for lung cancer therapy.

Unraveling the interplay between a small RNA and RNase E in bacteria.

Small RNAs (sRNAs) are major regulators of gene expression in bacteria, exerting their regulation primarily via base pairing with their target transcripts and modulating translation. Accumulating evidence suggest that sRNAs can also affect the stability of their target transcripts by altering their accessibility to endoribonucleases. Yet, the effects of sRNAs on transcript stability and the mechanisms underlying them have not been studied in wide scale. Here we employ large-scale RNA-seq-based methodologies in the model bacterium Escherichia coli to quantitatively study the functional interaction between a sRNA and an endoribonuclease in regulating gene expression, using the well-established sRNA, GcvB, and the major endoribonuclease, RNase E. Studying single and double mutants of gcvB and rne and analysing their RNA-seq results by the Double Mutant Cycle approach, we infer distinct modes of the interplay between GcvB and RNase E. Transcriptome-wide mapping of RNase E cleavage sites provides further support to the results of the RNA-seq analysis, identifying cleavage sites in targets in which the functional interaction between GcvB and RNase E is evident. Together, our results indicate that the most dominant mode of GcvB-RNase E functional interaction is GcvB enhancement of RNase E cleavage, which varies in its magnitude between different targets.

Human DDX6 regulates translation and decay of inefficiently translated mRNAs.

Recent findings indicate that the translation elongation rate influences mRNA stability. One of the factors that has been implicated in this link between mRNA decay and translation speed is the yeast DEAD-box helicase Dhh1p. Here, we demonstrated that the human ortholog of Dhh1p, DDX6, triggers the deadenylation-dependent decay of inefficiently translated mRNAs in human cells. DDX6 interacts with the ribosome through the Phe-Asp-Phe (FDF) motif in its RecA2 domain. Furthermore, RecA2-mediated interactions and ATPase activity are both required for DDX6 to destabilize inefficiently translated mRNAs. Using ribosome profiling and RNA sequencing, we identified two classes of endogenous mRNAs that are regulated in a DDX6-dependent manner. The identified targets are either translationally regulated or regulated at the steady-state-level and either exhibit signatures of poor overall translation or of locally reduced ribosome translocation rates. Transferring the identified sequence stretches into a reporter mRNA caused translation- and DDX6-dependent degradation of the reporter mRNA. In summary, these results identify DDX6 as a crucial regulator of mRNA translation and decay triggered by slow ribosome movement and provide insights into the mechanism by which DDX6 destabilizes inefficiently translated mRNAs.