Dataset Information

High Resolution N6-Methyladenosine (m6A) Map Using Photo-crosslinking-Assisted m6A Sequencing

ABSTRACT: We present here a photo-crosslinking-assisted m6A sequencing strategy to more accurately define sites with m6A modifications. Using this strategy, we obtained a high-resolution map of m6A in a human transcriptome. The map resembles the general distribution pattern observed previously, and reveals new m6A sites at base resolution. Our results provide insights into the relationship between the methylation regions and the binding sites of RNA-binding proteins.

ORGANISM(S): Homo sapiens

PROVIDER: GSE54921 | GEO | 2015/01/08

SECONDARY ACCESSION(S): PRJNA238038

REPOSITORIES: GEO

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Project description:Crosslinking mass spectrometry (XL-MS) is emerging as a unique method at the crossroads of structural and cellular biology, uniquely capable of identifying protein-protein interactions with residue-level resolution and on the proteome-wide scale. With the development of crosslinkers that can form linkages inside cells and easily cleave during fragmentation on the mass spectrometer (MS-cleavable crosslinks), it has become increasingly facile to identify contacts between any two proteins in complex samples, including in live cells or tissues. Photo-crosslinkers possess the advantages of high temporal resolution and high reactivity, thereby engaging all residue-types (rather than just lysine); nevertheless, photo-crosslinkers have not enjoyed widespread use, and have yet to be employed for proteome-wide studies, because their products are challenging to identify, and an MS-cleavable photo-crosslinker has not yet been reported. Here, we demonstrate the synthesis and application of two heterobifunctional photo-crosslinkers that feature diazirines and N-hydroxy-succinimidyl carbamate groups, the latter of which unveil MS-cleavable linkage upon acyl transfer to protein targets. Moreover, these crosslinkers demonstrate high water-solubility and cell-permeability. Using these compounds, we demonstrate the feasibility of proteome-wide photo-crosslinking mass spectrometry (photo-XL-MS), both in extracts and in cellulo. These studies provide a partial interaction map of the E. coli cytosol with residue-level resolution. We find that photo-XL-MS has a propensity to capture protein-protein interactions, particularly involving low-abundance uncharacterized proteins, suggesting it could be a powerful tool to shed light on the “darker” corners of the proteome. Overall, we describe methods that enable the detection of protein quinary interaction networks in their native environment at residue-level resolution proteome-wide, and we expect they will prove useful toward the effort to explore the molecular sociology of the cell.

Dataset Information

High Resolution N6-Methyladenosine (m6A) Map Using Photo-crosslinking-Assisted m6A Sequencing

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