Data@TAMU

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Browsing Data@TAMU by Funding Agency "DHHS-NIH-Eunice Kennedy Shriver National Institute of Child Health & Human Development"

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    Research Project
    CLAP-seq: An Aptamer-Based Platform for Transcriptome-Wide Mapping of RNA Modifications
    Chemistry; TAMU; https://hdl.handle.net/20.500.14641/200; DHHS-NIH-Eunice Kennedy Shriver National Institute of Child Health & Human Development
    Project Summary/Abstract Beginning in the 1950s, more than 100 types of posttranscriptional modifications have been identified in cellular RNA. Today, the study of RNA post-transcriptional modifications – known as epitranscriptomics – is a rapidly developing field, which promises to greatly enhance our understanding of human health and disease. Despite the profound implications already assigned to many RNA modifications, their precise functions remain poorly understood. This can be attributed to the lack of sensitive and robust sequencing technologies to detect these epitranscriptomics marks in a transcriptome-wide manner. A key bottleneck is the lack of sensitive and specific enrichment techniques (affinity- or reactivity-based) for RNA molecules containing these modifications. The proposed research takes direct aim at this critical deficit using the aptamer approach, employing in vitro selection methods to identify nucleic acid molecules that bind chemically modified RNAs. These aptamers are unique in that they are comprised of L-(deoxy)ribose-based nucleic acids (L-DNA and L-RNA), which are mirror images (enantiomers) of natural D-nucleotides. L-Aptamers, which are completely orthogonal to natural biology, are extremely well suited for binding RNA targets. Therefore, in vitro selection will be used to isolate novel L- aptamers capable of binding chemically modified mononucleotides, which will enable selective capture of RNA molecules containing the same modified residue. These L-aptamers will then be used in Cross-Linking- Aptamer Pull-down and sequencing (CLAP-seq), the first transcriptome-wide profiling technology employing aptamer-based RNA enrichment prior to next-generation sequencing. CLAP-seq not only promises to open a general and robust route towards transcriptome-wide profiling of the growing list of RNA modifications, but also promises to reinforce our current view of the epitranscriptome. Accordingly, the development of CLAP-seq will have a profound impact on the field of epitranscriptomics, which is well aligned with the mission of the NICHD and the goal of this FOA: to promote research into the role of RNA chemical modifications in development and related disease.
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    Research Project
    Global Changes In the 3UTRome Toggle Responsiveness To Growth Factors
    Ibt-Ctcr-Translational Cancer Research; TAMHSC; https://hdl.handle.net/20.500.14641/679; DHHS-NIH-Eunice Kennedy Shriver National Institute of Child Health & Human Development
    SUMMARY RNA binding proteins like the Pumilio family (PUFs) exert repression on 3'UTRs, and hence mRNA stability and translation. Alternative polyadenylation (APA) causes 3'UTR length to vary. Coordinated large-scale shortening of 3'UTRs through APA can lead to evasion from 3'UTR- mediated repressive signals and may be a key regulatory regime during development. Yet most identified cases of 3'UTR-mediated PUF repression - and APA to evade repression - were found ad hoc, between interacting gene pairs. Thus, a gap exists in our understanding of how these interactions are orchestrated at the global level of the 3'UTRome and APA. EGF patterns the C. elegans vulva. Of six equipotent vulval precursor cells (VPCs), the three closest to the EGF source are induced to form the vulva, while the distal three remain uninduced. Remarkably, this event occurs with 99.8% fidelity. Three redundant PUFs are expressed specifically in the uninduced cells, suggesting that distal VPCs enact a PUF- and 3'UTR-dependent program to become non-responsive to signal. In the germline, the same PUFs repress ERK/MAP kinase; this same mechanism may be adopted by non-responsive VPCs. Germline immunoprecipitation (IP) of one PUF identified many potential target 3'UTRs. From this dataset, we identified multiple target mRNAs from genes in each vulval signaling cascade (EGFR/Ras/Raf/MEK/ERK, Notch/CSL, PI3K/PDK/Akt, and EGFR/Ras/ RalGEF/Ral). We hypothesize that the redundant PUFs collectively repress mRNAs of all four identified signaling cascades to demarcate signal-non-responsive from signal-responsive cells. Our central hypothesis is that switching from proximal (short 3'UTR) to distal (long 3'UTR) polyadenylation sequence (PAS) usage governs switching from signal-responsiveness to non- responsiveness. We will systematically test this hypothesis by joining bottom-up and top-down specific aims. We propose to: 1) test 3'UTRs from the PUF IP list of vulval signaling genes for ability to mediate PUF-dependent reporter repression in non-responsive cells, 2) survey the VPC 3'UTRome and global proximal-to-distal PAS switching, and 3) validate select candidates by altering endogenous 3'UTRs and polyadenylation signals via CRISPR/Cas9 genome editing and deleting associated PUFs. We will ascertain the contribution to developmental fidelity by APA, and the changes in repressive access points in the 3'UTRs caused by APA.