RNA secondary structure dependence in METTL3–METTL14 mRNA methylation is modulated by the N-terminal domain of METTL3

AbstractN6-methyladenosine (m6A) is the most abundant modification in mRNA. The core of the human N6-methyltransferase complex (MTC) is formed by a heterodimer consisting of METTL3 and METTL14, which specifically catalyzes m6A formation within an RRACH sequence context. Using recombinant proteins in a site-specific methylation assay that allows determination of quantitative methylation yields, our results show that this complex methylates its target RNAs not only sequence but also secondary structure dependent. Furthermore, we demonstrate the role of specific protein domains on both RNA binding and substrate turnover, focusing on postulated RNA binding elements. Our results show that one zinc finger motif within the complex is sufficient to bind RNA, however, both zinc fingers are required for methylation activity. We show that the N-terminal domain of METTL3 alters the secondary structure dependence of methylation yields. Our results demonstrate that a cooperative effect of all RNA-binding elements in the METTL3–METTL14 complex is required for efficient catalysis, and that binding of further proteins affecting the NTD of METTL3 may regulate substrate specificity.

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RNA binding to human METTL3-METTL14 restricts N6-deoxyadenosine methylation of DNA in vitro

10.1101/2022.01.08.475504 ◽

2022 ◽

Author(s):

Shan Qi ◽

Javier Mota ◽

Siu-Hong Chan ◽

Johanna Villarreal ◽

Nan Dai ◽

...

Keyword(s):

Binding Affinity ◽

Rna Binding ◽

Methyl Group ◽

High Affinity ◽

Methylation Of Dna ◽

Dna And Rna ◽

Close Proximity ◽

Methylation Activity

Methyltransferase like-3 (METTL3) and METTL14 complex transfers a methyl group from S-adenosyl-L-methionine to N6 amino group of adenosine bases in RNA (m6A) and DNA (m6dA). Emerging evidence highlights a role of METTL3-METTL14 in the chromatin context, especially in processes where DNA and RNA are held in close proximity. However, a mechanistic framework about specificity for substrate RNA/DNA and their interrelationship remain unclear. By systematically studying methylation activity and binding affinity to a number of DNA and RNA oligos with different propensities to form inter- or intra-molecular duplexes or single-stranded molecules in vitro, we uncover an inverse relationship for substrate binding and methylation and show that METTL3-METTL14 preferentially catalyzes the formation of m6dA in single-stranded DNA (ssDNA), despite weaker binding affinity to DNA. In contrast, it binds structured RNAs with high affinity, but methylates the target adenosine in RNA (m6A) much less efficiently than it does in ssDNA. We also show that METTL3-METTL14-mediated methylation of DNA is largely restricted by structured RNA elements prevalent in long noncoding and other cellular RNAs.

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Systematic Identification of Protein Phosphorylation-Mediated Interactions

10.1101/2020.09.18.304121 ◽

2020 ◽

Author(s):

Brendan M. Floyd ◽

Kevin Drew ◽

Edward M. Marcotte

Keyword(s):

Mass Spectrometry ◽

Protein Phosphorylation ◽

Protein Interactions ◽

Rna Binding ◽

Rna Binding Proteins ◽

Specific Protein ◽

Mass Spectrometry Data ◽

Size Exclusion ◽

Functional Protein

ABSTRACTProtein phosphorylation is a key regulatory mechanism involved in nearly every eukaryotic cellular process. Increasingly sensitive mass spectrometry approaches have identified hundreds of thousands of phosphorylation sites but the functions of a vast majority of these sites remain unknown, with fewer than 5% of sites currently assigned a function. To increase our understanding of functional protein phosphorylation we developed an approach for identifying the phosphorylation-dependence of protein assemblies in a systematic manner. A combination of non-specific protein phosphatase treatment, size-exclusion chromatography, and mass spectrometry allowed us to identify changes in protein interactions after the removal of phosphate modifications. With this approach we were able to identify 316 proteins involved in phosphorylation-sensitive interactions. We recovered known phosphorylation-dependent interactors such as the FACT complex and spliceosome, as well as identified novel interactions such as the tripeptidyl peptidase TPP2 and the supraspliceosome component ZRANB2. More generally, we find phosphorylation-dependent interactors to be strongly enriched for RNA-binding proteins, providing new insight into the role of phosphorylation in RNA binding. By searching directly for phosphorylated amino acid residues in mass spectrometry data, we identified the likely regulatory phosphosites on ZRANB2 and FACT complex subunit SSRP1. This study provides both a method and resource for obtaining a better understanding of the role of phosphorylation in native macromolecular assemblies.

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Determination of the Secondary Structure and Folding Topology of an RNA Binding Domain of Mammalian hnRNP A1 Protein Using Three-Dimensional Heteronuclear Magnetic Resonance Spectroscopy

Biochemistry ◽

10.1021/bi00176a015 ◽

1994 ◽

Vol 33 (10) ◽

pp. 2852-2858 ◽

Cited By ~ 26

Author(s):

Daniel S. Garrett ◽

Patricia J. Lodi ◽

Yousif Shamoo ◽

Kenneth R. Williams ◽

G. Marius Clore ◽

...

Keyword(s):

Magnetic Resonance ◽

Secondary Structure ◽

Magnetic Resonance Spectroscopy ◽

Rna Binding ◽

Three Dimensional ◽

Binding Domain ◽

Resonance Spectroscopy ◽

Hnrnp A1 ◽

Rna Binding Domain

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Determination of the structural role of the N-terminal domain of human extracellular superoxide dismutase by use of protein fusions

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology ◽

10.1016/0167-4838(95)00189-1 ◽

1996 ◽

Vol 1292 (1) ◽

pp. 47-52 ◽

Cited By ~ 28

Author(s):

Lena A.E. Tibell ◽

Eleonore Skärfstad ◽

Bengt-Harald Jonsson

Keyword(s):

Superoxide Dismutase ◽

Extracellular Superoxide Dismutase ◽

Structural Role ◽

Terminal Domain

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Translation of the psbA mRNA of Chlamydomonas reinhardtii requires a structured RNA element contained within the 5' untranslated region.

The Journal of Cell Biology ◽

10.1083/jcb.127.6.1537 ◽

1994 ◽

Vol 127 (6) ◽

pp. 1537-1545 ◽

Cited By ~ 74

Author(s):

S P Mayfield ◽

A Cohen ◽

A Danon ◽

C B Yohn

Keyword(s):

Secondary Structure ◽

Untranslated Region ◽

Translational Regulation ◽

Rna Binding ◽

Rna Binding Proteins ◽

Higher Plants ◽

Consensus Sequence ◽

Specific Protein ◽

Rna Elements ◽

Ribosome Association

Translational regulation is a key modulator of gene expression in chloroplasts of higher plants and algae. Genetic analysis has shown that translation of chloroplast mRNAs requires nuclear-encoded factors that interact with chloroplastic mRNAs in a message-specific manner. Using site-specific mutations of the chloroplastic psbA mRNA, we show that RNA elements contained within the 5' untranslated region of the mRNA are required for translation. One of these elements is a Shine-Dalgarno consensus sequence, which is necessary for ribosome association and psbA translation. A second element required for high levels of psbA translation is located adjacent to and upstream of the Shine-Dalgarno sequence, and maps to the location on the RNA previously identified as the site of message-specific protein binding. This second element appears to act as a translational attenuator that must be overcome to activate translation. Mutations that affect the secondary structure of these RNA elements greatly reduce the level of psbA translation, suggesting that secondary structure of these RNA elements plays a role in psbA translation. These data suggest a mechanism for translational activation of the chloroplast psbA mRNA in which an RNA element containing the ribosome-binding site is bound by message-specific RNA binding proteins allowing for increased ribosome association and translation initiation. These elements may be involved in the light-regulated translation of the psbA mRNA.

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Role of the Acidic Carboxyl-terminal Domain of the Single-stranded DNA-binding Protein of Bacteriophage T7 in Specific Protein-Protein Interactions

Journal of Biological Chemistry ◽

10.1074/jbc.273.11.6556 ◽

1998 ◽

Vol 273 (11) ◽

pp. 6556-6564 ◽

Cited By ~ 31

Author(s):

Daochun Kong ◽

Charles C. Richardson

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

Binding Protein ◽

Dna Binding Protein ◽

Specific Protein ◽

Bacteriophage T7 ◽

Protein Protein Interactions ◽

Terminal Domain ◽

Carboxyl Terminal

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Assignments of 1H, 15N, and 13C Resonances for the Backbone and Side Chains of the N-Terminal Domain of DNA Polymerase .beta.. Determination of the Secondary Structure and Tertiary Contacts

Biochemistry ◽

10.1021/bi00198a020 ◽

1994 ◽

Vol 33 (32) ◽

pp. 9537-9545 ◽

Cited By ~ 16

Author(s):

Dingjiang Liu ◽

Eugene F. DeRose ◽

Rajendra Prasad ◽

Samuel H. Wilson ◽

Gregory P. Mullen

Keyword(s):

Secondary Structure ◽

Dna Polymerase ◽

Dna Polymerase Beta ◽

Side Chains ◽

Polymerase Beta ◽

Terminal Domain ◽

Tertiary Contacts

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LncRNA MIR99AHG mediated by FOXA1 modulates NOTCH2/Notch signaling pathway to accelerate pancreatic cancer through sponging miR-3129-5p and recruiting ELAVL1

Cancer Cell International ◽

10.1186/s12935-021-02189-z ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Jin Xu ◽

Weixue Xu ◽

Xuan Yang ◽

Zhen Liu ◽

Yiya Zhao ◽

...

Keyword(s):

Pancreatic Cancer ◽

Notch Signaling ◽

Signaling Pathway ◽

Rna Binding ◽

Malignant Tumors ◽

Notch Signaling Pathway ◽

Notch Receptor ◽

Migration And Invasion

Abstract Background Pancreatic cancer (PCa) is a fatal malignancy with poor prognosis, high recurrence and mortality. Substantial reports have suggested long non-coding RNAs (lncRNAs) are implicated in development of numerous malignant tumors, and PCa is included. However, the correlation between novel lncRNA mir-99a-let-7c cluster host gene (MIR99AHG) and PCa remains elusive and needs to be deeply investigated. Methods In this study, we firstly used RT-qPCR to examine MIR99AHG expression. Functional assays were implemented for determination of the role of MIR99AHG in PCa cells. Mechanism experiments were designed and carried out for exploring the regulatory mechanism involving MIR99AHG. Results MIR99AHG was distinctly overexpressed in PCa cell lines. MIR99AHG deficiency abrogated PCa cell proliferation, migration and invasion. Moreover, MIR99AHG up-regulation was induced by transcription factor forkhead box A1 (FOXA1). Furthermore, MIR99AHG modulated notch receptor 2 (NOTCH2) expression and stimulated Notch signaling pathway through sequestering microRNA-3129-5p (miR-3129-5p) and recruiting ELAV like RNA binding protein 1 (ELAVL1). Conclusions Altogether, the exploration of FOXA1/MIR99AHG/miR-3129-5p/ELAVL1/NOTCH2 axis in the progression of PCa might provide a meaningful revelation for PCa diagnosis and treatment.

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Inhibition of Protein Synthesis by Y Box-Binding Protein 1 Blocks Oncogenic Cell Transformation

Molecular and Cellular Biology ◽

10.1128/mcb.25.6.2095-2106.2005 ◽

2005 ◽

Vol 25 (6) ◽

pp. 2095-2106 ◽

Cited By ~ 53

Author(s):

Andreas G. Bader ◽

Peter K. Vogt

Keyword(s):

Rna Binding ◽

Mutational Analysis ◽

Cell Transformation ◽

Binding Protein ◽

Pi3k Pathway ◽

Terminal Domain ◽

Localization Signal ◽

Phosphoinositide 3 Kinase ◽

Mrna Binding

ABSTRACT The multifunctional Y box-binding protein 1 (YB-1) is transcriptionally repressed by the oncogenic phosphoinositide 3-kinase (PI3K) pathway (with P3K as an oncogenic homolog of the catalytic subunit) and, when reexpressed with the retroviral vector RCAS, interferes with P3K- and Akt-induced transformation of chicken embryo fibroblasts. Retrovirally expressed YB-1 binds to the cap of mRNAs and inhibits cap-dependent and cap-independent translation. To determine the requirements for the inhibitory role of YB-1 in P3K-induced transformation, we conducted a mutational analysis, measuring YB-1-induced interference with transformation, subcellular localization, cap binding, mRNA binding, homodimerization, and inhibition of translation. The results show that (i) interference with transformation requires RNA binding and a C-terminal domain that is distinct from the cytoplasmic retention domain, (ii) interference with transformation is tightly correlated with inhibition of translation, and (iii) masking of mRNAs by YB-1 is not sufficient to block transformation or to inhibit translation. We identified a noncanonical nuclear localization signal (NLS) in the C-terminal half of YB-1. A mutant lacking the NLS retains its ability to interfere with transformation, indicating that a nuclear function is not required. These results suggest that YB-1 interferes with P3K-induced transformation by a specific inhibition of translation through its RNA-binding domain and a region in the C-terminal domain. Potential functions of the C-terminal region are discussed.

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