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MRNA psi profiling using nanopore DRS reveals cell type-specific pseudouridylation

ABSTRACT: Pseudouridine (psi) is one of the most abundant human mRNA modifications generated via psi synthases, including TRUB1 and PUS7. Nanopore direct RNA sequencing combined with our recent tool, Mod-p ID, enables psi mapping, transcriptome-wide, without chemical derivatization of the input RNA and/or conversion to cDNA. This method is sensitive for detecting changes in positional psi occupancies across cell types, which can inform our understanding of the impact on gene expression. We sequenced, mapped, and compared the positional psi occupancy across six immortalized human cell lines derived from diverse tissue types. We found that lung-derived cells have the highest proportion of psi, while liver-derived cells have the lowest. Further, we find that conserved psi positions correspond to higher levels of protein expression than expected, suggesting translation regulation. Interestingly, we identify cell type-specific sites of psi modification in ubiquitously expressed genes. Finally, we characterize sites with multiple psi modifications on the same transcript and found that these can be conserved or cell type specific, including examples of multiple psi modifications within the same k-mer. Our data support the hypothesis that psi modifications contribute to regulating translation and that cell type-specific trans-acting factors play a major role in driving pseudouridylation.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Sara Rouhanifard

PROVIDER: MSV000095673 | MassIVE | Thu Aug 22 09:22:00 BST 2024

SECONDARY ACCESSION(S): PXD055082

REPOSITORIES: MassIVE

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Project description:Extensive changes in replication timing occur during early mouse development, but their biological significance remains uncertain. To identify evolutionarily conserved features of replication timing and their relationships to epigenetic properties in humans, we profiled replication timing genome-wide in four human embryonic stem cell (hESC) lines, hESC-derived neural precursor cells (NPCs), lymphoblastoid cells, and two independently derived human induced pluripotent stem cell lines (hiPSCs). Results confirm the conservation of coordinately replicated megabase-sized units of chromosomes (replication domains) with stable cell type specific molecular boundaries that consolidate into larger replication domains during differentiation. Replication timing changes encompassed units of 400-800 kb and were coordinated with changes in transcription similar to mouse. Moreover, significant cell-type specific conservation of replication timing profiles was observed across regions of conserved synteny, despite significant species variation in the alignment of replication timing to isochore GC/LINE-1 content. Replication profiling also revealed a closer genome-wide epigenetic alignment of hESCs to mouse epiblast-derived stem cells (mEpiSCs) than to mouse ESCs. Finally, we identify a signature of chromatin modifications marking the boundaries of early replicating domains and a remarkably strong link between spatial proximity of chromatin as measured by Hi-C analysis and replication timing. Together, our results reveal evolutionarily conserved elements of the replication program in mammalian early development, demonstrate the power of replication profiling to identify important epigenetic distinctions between closely related stem cell populations (e.g. ESCs vs. EpiSCs), and strengthen the hypothesis that replication domains are structural and functional units of 3D chromosomal architecture. 8 cell types, with a total of 13 individual replicates (i.e. 5 in duplicates, 3 in single replicates)

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MRNA psi profiling using nanopore DRS reveals cell type-specific pseudouridylation

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