The long non-coding RNA X-inactive specific transcript (XIST) mediates the transcriptional silencing of genes on the X chromosome. Here we show that, in human cells, XIST is highly methylated with at ...least 78 N
-methyladenosine (m
A) residues-a reversible base modification of unknown function in long non-coding RNAs. We show that m
A formation in XIST, as well as in cellular mRNAs, is mediated by RNA-binding motif protein 15 (RBM15) and its paralogue RBM15B, which bind the m
A-methylation complex and recruit it to specific sites in RNA. This results in the methylation of adenosine nucleotides in adjacent m
A consensus motifs. Furthermore, we show that knockdown of RBM15 and RBM15B, or knockdown of methyltransferase like 3 (METTL3), an m
A methyltransferase, impairs XIST-mediated gene silencing. A systematic comparison of m
A-binding proteins shows that YTH domain containing 1 (YTHDC1) preferentially recognizes m
A residues on XIST and is required for XIST function. Additionally, artificial tethering of YTHDC1 to XIST rescues XIST-mediated silencing upon loss of m
A. These data reveal a pathway of m
A formation and recognition required for XIST-mediated transcriptional repression.
N
-methyladenosine (m
A) is an abundant nucleotide modification in mRNA that is required for the differentiation of mouse embryonic stem cells. However, it remains unknown whether the m
A ...modification controls the differentiation of normal and/or malignant myeloid hematopoietic cells. Here we show that shRNA-mediated depletion of the m
A-forming enzyme METTL3 in human hematopoietic stem/progenitor cells (HSPCs) promotes cell differentiation, coupled with reduced cell proliferation. Conversely, overexpression of wild-type METTL3, but not of a catalytically inactive form of METTL3, inhibits cell differentiation and increases cell growth. METTL3 mRNA and protein are expressed more abundantly in acute myeloid leukemia (AML) cells than in healthy HSPCs or other types of tumor cells. Furthermore, METTL3 depletion in human myeloid leukemia cell lines induces cell differentiation and apoptosis and delays leukemia progression in recipient mice in vivo. Single-nucleotide-resolution mapping of m
A coupled with ribosome profiling reveals that m
A promotes the translation of c-MYC, BCL2 and PTEN mRNAs in the human acute myeloid leukemia MOLM-13 cell line. Moreover, loss of METTL3 leads to increased levels of phosphorylated AKT, which contributes to the differentiation-promoting effects of METTL3 depletion. Overall, these results provide a rationale for the therapeutic targeting of METTL3 in myeloid leukemia.
N
-Methyladenosine (m
A) on mRNAs mediates different biological processes and its dysregulation contributes to tumorigenesis. How m
A dictates its diverse molecular and cellular effects in leukemias ...remains unknown. We found that YTHDC1 is the essential m
A reader in myeloid leukemia from a genome-wide CRISPR screen and that m
A is required for YTHDC1 to undergo liquid-liquid phase separation and form nuclear YTHDC1-m
A condensates (nYACs). The number of nYACs increases in acute myeloid leukemia (AML) cells compared with normal hematopoietic stem and progenitor cells. AML cells require the nYACs to maintain cell survival and the undifferentiated state that is critical for leukemia maintenance. Furthermore, nYACs enable YTHDC1 to protect m
A-mRNAs from the PAXT complex and exosome-associated RNA degradation. Collectively, m
A is required for the formation of a nuclear body mediated by phase separation that maintains mRNA stability and control cancer cell survival and differentiation.
N
6
-methyladenosine (m
6
A) is a highly prevalent mRNA modification that promotes degradation of transcripts encoding proteins that have roles in cell development, differentiation, and other ...pathways. METTL3 is the major methyltransferase that catalyzes the formation of m
6
A in mRNA. As 30% to 80% of m
6
A can remain in mRNA after METTL3 depletion by CRISPR/Cas9-based methods, other enzymes are thought to catalyze a sizable fraction of m
6
A. Here, we reexamined the source of m
6
A in the mRNA transcriptome. We characterized mouse embryonic stem cell lines that continue to have m
6
A in their mRNA after
Mettl3
knockout. We show that these cells express alternatively spliced
Mettl3
transcript isoforms that bypass the CRISPR/Cas9 mutations and produce functionally active methyltransferases. We similarly show that other reported
METTL3
knockout cell lines express altered METTL3 proteins. We find that gene dependency datasets show that most cell lines fail to proliferate after
METTL3
deletion, suggesting that reported
METTL3
knockout cell lines express altered METTL3 proteins rather than have full knockout. Finally, we reassessed METTL3’s role in synthesizing m
6
A using an exon 4 deletion of
Mettl3
and found that METTL3 is responsible for >95% of m
6
A in mRNA. Overall, these studies suggest that METTL3 is responsible for the vast majority of m
6
A in the transcriptome, and that remaining m
6
A in putative
METTL3
knockout cell lines is due to the expression of altered but functional METTL3 isoforms.
In Escherichia coli, cytokinesis is orchestrated by FtsZ, which forms a Z‐ring to drive septation. Spatial and temporal control of Z‐ring formation is achieved by the Min and nucleoid occlusion (NO) ...systems. Unlike the well‐studied Min system, less is known about the anti‐DNA guillotining NO process. Here, we describe studies addressing the molecular mechanism of SlmA (synthetic lethal with a defective Min system)‐mediated NO. SlmA contains a TetR‐like DNA‐binding fold, and chromatin immunoprecipitation analyses show that SlmA‐binding sites are dispersed on the chromosome except the Ter region, which segregates immediately before septation. SlmA binds DNA and FtsZ simultaneously, and the SlmA–FtsZ structure reveals that two FtsZ molecules sandwich a SlmA dimer. In this complex, FtsZ can still bind GTP and form protofilaments, but the separated protofilaments are forced into an anti‐parallel arrangement. This suggests that SlmA may alter FtsZ polymer assembly. Indeed, electron microscopy data, showing that SlmA–DNA disrupts the formation of normal FtsZ polymers and induces distinct spiral structures, supports this. Thus, the combined data reveal how SlmA derails Z‐ring formation at the correct place and time to effect NO.
Nucleoid occlusion (NO) restricts bacterial cell division to prevent chromosome guillotining in the cell midzone when replication or segregation is delayed. Structural work suggests that the NO factor SlmA (synthetic lethal with a defective Min system) interferes with formation of the cytokinetic Z‐ring by altering associations between FtsZ protofilaments.
MicroRNAs (miRNA) are endogenous, short, non-coding RNA that undergo a multistep biogenesis before generating the functional, mature sequence. The core components of the microprocessor complex, ...consisting of Drosha and DGCR8, are both necessary and sufficient for this process, although accessory proteins have been found that modulate the biogenesis of a subset of miRNA. Curiously, many of the proteins involved in miRNA biogenesis are also needed for ribosomal RNA processing. Here we show that nucleolin, another protein critical for rRNA processing, is involved in the biogenesis of microRNA 15a/16 (miR-15a/16), specifically at the primary to precursor stage of processing. Through overexpression and knockdown studies, we show that miR-15a/16 levels are directly correlated to nucleolin expression. Furthermore, we found that cellular localization is critical for the proper functioning of nucleolin in this pathway and that nucleolin directly interacts with DGCR8 and Drosha in the nucleus. Nucleolin can bind to the primary miRNA both directly and specifically. Finally, we show that in the absence of nucleolin, cell extracts are unable to process miR-15a/16 in vitro and that this can be rescued by the addition of nucleolin. Our findings offer a new protein component in the microRNA biogenesis pathway and lend insight into miRNA dysregulation in certain cancers.
Background: MicroRNA processing is a tightly controlled multistep process involving accessory proteins.
Results: Expression of microRNAs 15a and 16 is controlled by nucleolin expression and localization.
Conclusion: Nucleolin facilitates the biogenesis of microRNAs 15a and 16 through direct interaction with the microprocessor complex.
Significance: Nucleolin represents a novel component of the microRNA processing pathway.
N6-Methyladenosine (m6A) is the most prevalent post-transcriptional modification of eukaryotic mRNA and long noncoding RNA. m6A mediates its effects primarily by recruiting proteins, including the ...multiprotein eukaryotic initiation factor 3 complex and a set of proteins that contain the YTH domain. Here we describe the mechanisms by which YTH domain-containing proteins bind m6A and influence the fate of m6A-containing RNA in mammalian cells. We discuss the diverse, and occasionally contradictory, functions ascribed to these proteins and the emerging concepts that are influencing our understanding of these proteins and their effects on the epitranscriptome.
m6A is the most abundant mRNA modification. The YTH domain specifically recognizes m6A and is conserved from yeast to humans.
The first function assigned to a YTH protein was destabilization of mRNAs by YTHDF2. Since then YTH proteins have been linked to both mRNA degradation and protein translation (YTHDF1 and YTHDF3), and regulate splicing and chromatin modification (YTHDC1).
m6A has been mapped in multiple viruses and binding of YTH proteins was shown to either help or inhibit viral replication. Nuclear replicating viruses are helped by YTH proteins, while cytoplasmic replicating viruses are inhibited.
Dysregulated mTORC1 signaling alters a wide range of cellular processes, contributing to metabolic disorders and cancer. Defining the molecular details of downstream effectors is thus critical for ...uncovering selective therapeutic targets. We report that mTORC1 and its downstream kinase S6K enhance eIF4A/4B-mediated translation of Wilms’ tumor 1-associated protein (WTAP), an adaptor for the N6-methyladenosine (m6A) RNA methyltransferase complex. This regulation is mediated by 5′ UTR of WTAP mRNA that is targeted by eIF4A/4B. Single-nucleotide-resolution m6A mapping revealed that MAX dimerization protein 2 (MXD2) mRNA contains m6A, and increased m6A modification enhances its degradation. WTAP induces cMyc-MAX association by suppressing MXD2 expression, which promotes cMyc transcriptional activity and proliferation of mTORC1-activated cancer cells. These results elucidate a mechanism whereby mTORC1 stimulates oncogenic signaling via m6A RNA modification and illuminates the WTAP-MXD2-cMyc axis as a potential therapeutic target for mTORC1-driven cancers.
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•mTORC1 induces WTAP translation through eIF4A/4B•mTORC1-WTAP increases global m6A levels on mRNA•Increased m6A modification by mTORC1 destabilizes mRNAs•mTORC1 decreases mRNA expression of MXD2, cMyc suppressor, through m6A modification
Cho et al. report that mTORC1 enhances WTAP translation through eIF4A/4B, resulting in the increase of global mRNA m6A modification. m6A modification of mRNAs by mTORC1 destabilizes transcripts through YTHDF m6A-reader proteins. Among the targets, mTORC1 increases m6A modification on MXD2 mRNA, cMyc suppressor, resulting in increased cMyc activity and tumor growth.
N6-Methyladenosine (m6A) on mRNAs mediates different biological processes and its dysregulation contributes to tumorigenesis. How m6A dictates its diverse molecular and cellular effects in leukemias ...remains unknown. We found that YTHDC1 is the essential m6A reader in myeloid leukemia from a genome-wide CRISPR screen and that m6A is required for YTHDC1 to undergo liquid-liquid phase separation and form nuclear YTHDC1-m6A condensates (nYACs). The number of nYACs increases in acute myeloid leukemia (AML) cells compared with normal hematopoietic stem and progenitor cells. AML cells require the nYACs to maintain cell survival and the undifferentiated state that is critical for leukemia maintenance. Furthermore, nYACs enable YTHDC1 to protect m6A-mRNAs from the PAXT complex and exosome-associated RNA degradation. Collectively, m6A is required for the formation of a nuclear body mediated by phase separation that maintains mRNA stability and control cancer cell survival and differentiation.
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•YTHDC1 is required for AML cell survival, differentiation state, and leukemogenesis•YTHDC1 binds to m6A and forms nuclear condensates (nYACs) mediated by LLPS•nYACs are more abundant in AML cells compared with normal blood cells•nYACs protect mRNAs (i.e., MYC and others) from degradation by the PAXT complex
Using AML cell lines and patient samples Cheng et al. identify a requirement for YTHDC1 in myeloid leukemogenesis. YTHDC1 undergoes liquid-liquid phase separation by binding to m6A to form dynamic nuclear condensates. These nuclear bodies are increased in myeloid leukemia cells and protect mRNAs from the PAXT-exosome complex.
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