METTL3-STAT5B interaction facilitates the co-transcriptional m6A modification of mRNA to promote breast tumorigenesis.

Enhanced expression of methyltransferase-like 3 (METTL3) promotes the m6A modification of specific mRNAs, contributing to breast tumorigenesis. While the mRNA substrates targeted by METTL3 are well characterized, the factors dictating the selection of these specific mRNA remain elusive. This study aimed to examine the regulatory role of the transcription factor STAT5B in METTL3-induced m6A modification. METTL3 specifically interacts with STAT5B in response to mitogenic stimulation by epidermal growth factor (EGF). Chromatin immunoprecipitation and CRISPR/Cas9 mutagenesis showed that STAT5B recruits METTL3 to gene promoters like CCND1, where METTL3 interacts with RPB1, dependent on CDK9-mediated RPB1 (Ser2) phosphorylation during transcription elongation. Inhibition and depletion of either STAT5B or CDK9 prevented the EGF-induced m6A modification of CCND1. The translation efficiency of CCND1 was increased following m6A modification, thereby increasing cell proliferation. STAT5B facilitated METTL3-induced tumor formation by increasing CCND1 expression in an orthotopic mouse model. In clinical context, a positive correlation was observed between p-STAT5B and METTL3 expression in high-grade breast tumors. This study elucidates a novel mechanism that underlies the specificity of m6A modification in breast cancer cells, thereby underscoring its potential therapeutic value.

Hematopoietic stem cell niche generation and maintenance are distinguishable by an epitranscriptomic program.

The niche is typically considered as a pre-established structure sustaining stem cells. Therefore, the regulation of its formation remains largely unexplored. Whether distinct molecular mechanisms control the establishment versus maintenance of a stem cell niche is unknown. To address this, we compared perinatal and adult bone marrow mesenchymal stromal cells (MSCs), a key component of the hematopoietic stem cell (HSC) niche. MSCs exhibited enrichment in genes mediating m6A mRNA methylation at the perinatal stage and downregulated the expression of Mettl3, the m6A methyltransferase, shortly after birth. Deletion of Mettl3 from developing MSCs but not osteoblasts led to excessive osteogenic differentiation and a severe HSC niche formation defect, which was significantly rescued by deletion of Klf2, an m6A target. In contrast, deletion of Mettl3 from MSCs postnatally did not affect HSC niche. Stem cell niche generation and maintenance thus depend on divergent molecular mechanisms, which may be exploited for regenerative medicine.

Mettl14-mediated m6A modification ensures the cell-cycle progression of late-born retinal progenitor cells.

Neural progenitor cells lengthen their cell cycle to prime themselves for differentiation as development proceeds. It is currently not clear how they counter this lengthening and avoid being halted in the cell cycle. We show that N6-methyladenosine (m6A) methylation of cell-cycle-related mRNAs ensures the proper cell-cycle progression of late-born retinal progenitor cells (RPCs), which are born toward the end of retinogenesis and have long cell-cycle length. Conditional deletion of Mettl14, which is required for depositing m6A, led to delayed cell-cycle exit of late-born RPCs but has no effect on retinal development prior to birth. m6A sequencing and single-cell transcriptomics revealed that mRNAs involved in elongating the cell cycle were highly enriched for m6A, which could target them for degradation and guarantee proper cell-cycle progression. In addition, we identified Zfp292 as a target of m6A and potent inhibitor of RPC cell-cycle progression.

Ribosomal RNA-based epitranscriptomic regulation of chondrocyte translation and proteome in osteoarthritis.

OBJECTIVE: Osteoarthritis-related cartilage extracellular matrix remodeling is dependent on changes in chondrocyte protein expression. Yet, the role of ribosomes in chondrocyte translation regulation is unknown. In this exploratory study, we investigated ribosomal RNA (rRNA) epitranscriptomic-based ribosome heterogeneity in human articular chondrocytes and its relevance for osteoarthritis. METHODS: Sequencing-based rRNA 2'-O-methylation profiling analysis (RiboMethSeq) was performed on non-OA primary human articular chondrocytes (n = 5) exposed for 14 days to osteoarthritic synovial fluid (14 donors, pooled, 20% v/v). The SW1353 SNORD71 KO cell pool was generated using LentiCRISPRv2/Cas9. The mode of translation initiation and fidelity were determined by dual-luciferase reporters. The cellular proteome was analyzed by LC-MS/MS and collagen type I protein expression was evaluated by immunoblotting. Loading of COL1A1 mRNA into polysomes was determined by sucrose gradient ultracentrifugation and fractionation. RESULTS: We discovered that osteoarthritic synovial fluid instigates site-specific changes in the rRNA 2'-O-me profile of primary human articular chondrocytes. We identified five sites with differential 2'-O-me levels. The 2'-O-me status of 5.8S-U14 (one of identified differential 2'-O-me sites; decreased by 7.7%, 95% CI [0.9-14.5%]) was targeted by depleting the level of its guide snoRNA SNORD71 (50% decrease, 95% CI [33-64%]). This resulted in an altered ribosome translation modus (e.g., CrPV IRES, FC 3, 95% CI [2.2-4.1]) and promoted translation of COL1A1 mRNA which led to increased levels of COL1A1 protein (FC 1.7, 95% CI [1.3-2.0]). CONCLUSIONS: Our data identify a novel concept suggesting that articular chondrocytes employ rRNA epitranscriptomic mechanisms in osteoarthritis development.

Control of animal virus replication by RNA adenosine methylation.

Methylation at the N6-position of either adenosine (m6A) or 2'-O-methyladenosine (m6Am) represents two of the most abundant internal modifications of coding and non-coding RNAs, influencing their maturation, stability and function. Additionally, although less abundant and less well-studied, monomethylation at the N1-position (m1A) can have profound effects on RNA folding. It has been known for several decades that RNAs produced by both DNA and RNA viruses can be m6A/m6Am modified and the list continues to broaden through advances in detection technologies and identification of the relevant methyltransferases. Recent studies have uncovered varied mechanisms used by viruses to manipulate the m6A pathway in particular, either to enhance virus replication or to antagonize host antiviral defenses. As such, RNA modifications represent an important frontier of exploration in the broader realm of virus-host interactions, and this new knowledge already suggests exciting opportunities for therapeutic intervention. In this review we summarize the principal mechanisms by which m6A/m6Am can promote or hinder viral replication, describe how the pathway is actively manipulated by biomedically important viruses, and highlight some remaining gaps in understanding how adenosine methylation of RNA controls viral replication and pathogenesis.

The Landscape of IFN/ISG Signaling in HIV-1-Infected Macrophages and Its Possible Role in the HIV-1 Latency.

A key characteristic of Human immunodeficiency virus type 1 (HIV-1) infection is the generation of latent viral reservoirs, which have been associated with chronic immune activation and sustained inflammation. Macrophages play a protagonist role in this context since they are persistently infected while being a major effector of the innate immune response through the generation of type-I interferons (type I IFN) and IFN-stimulated genes (ISGs). The balance in the IFN signaling and the ISG induction is critical to promote a successful HIV-1 infection. Classically, the IFNs response is fine-tuned by opposing promotive and suppressive signals. In this context, it was described that HIV-1-infected macrophages can also synthesize some antiviral effector ISGs and, positive and negative regulators of the IFN/ISG signaling. Recently, epitranscriptomic regulatory mechanisms were described, being the N6-methylation (m6A) modification on mRNAs one of the most relevant. The epitranscriptomic regulation can affect not only IFN/ISG signaling, but also type I IFN expression, and viral fitness through modifications to HIV-1 RNA. Thus, the establishment of replication-competent latent HIV-1 infected macrophages may be due to non-classical mechanisms of type I IFN that modulate the activation of the IFN/ISG signaling network.

Epitranscriptomic addition of m6A regulates HIV-1 RNA stability and alternative splicing.

Previous work has demonstrated that the epitranscriptomic addition of m6A to viral transcripts can promote the replication and pathogenicity of a wide range of DNA and RNA viruses, including HIV-1, yet the underlying mechanisms responsible for this effect have remained unclear. It is known that m6A function is largely mediated by cellular m6A binding proteins or readers, yet how these regulate viral gene expression in general, and HIV-1 gene expression in particular, has been controversial. Here, we confirm that m6A addition indeed regulates HIV-1 RNA expression and demonstrate that this effect is largely mediated by the nuclear m6A reader YTHDC1 and the cytoplasmic m6A reader YTHDF2. Both YTHDC1 and YTHDF2 bind to multiple distinct and overlapping sites on the HIV-1 RNA genome, with YTHDC1 recruitment serving to regulate the alternative splicing of HIV-1 RNAs. Unexpectedly, while YTHDF2 binding to m6A residues present on cellular mRNAs resulted in their destabilization as previously reported, YTHDF2 binding to m6A sites on HIV-1 transcripts resulted in a marked increase in the stability of these viral RNAs. Thus, YTHDF2 binding can exert diametrically opposite effects on RNA stability, depending on RNA sequence context.