mRNA stability varies considerably from one mRNA species to another and plays an important role in determining levels of gene expression. Differential mRNA decay rates are determined by specific cis-acting elements within the mRNA molecule. The AU-rich element (ARE) is the most common cis element responsible for rapid mRNA decay in mammalian cells and can be found in the 3UTRs of short-lived transcripts encoding cytokines, chemokines, transcription factors, proto- oncogenes, and cell-cycle regulators. It is now clear that AREs may account for the degradation of most unstable mRNAs and that the regulation of mRNA half-life plays a crucial role in the control of gene expression. Numerous proteins have been described to bind AREs (ARE-binding protein (ARE-BP). Some are mRNA decay-promoting factors while others are stabilizing factors. In addition, the function of some of these proteins in destabilizing or stabilizing mRNAs is dependent on the cellular context and/or the expressed protein isoforms. We first focused on identifying the target mRNA binding sites of three ARE-BPs, ELAVL1/HuR, ZFP36/TTP, and HNRNPD/AUF1, known to be involved in clearance of short half-life messages. We found that targets bound and negatively regulated by ZFP36 included transcripts encoding proteins necessary for immune function and cancer, and transcripts encoding other RBPs. Genes with increased mRNA half-lives in ZFP36 knockout versus wild-type mouse cells were significantly enriched for our human ZFP36 targets. We identified thousands of overlapping ZFP36 and ELAVL1 binding sites, in 1,313 genes, and found that ZFP36 degrades transcripts through specific AU-rich sequences, representing a subset of the U-rich sequences ELAVL1 interacts with to stabilize transcripts. In a related study dissecting the regulatory effects of HNRNPD we found, as anticipated based on studies on individual target transcript stability, that HNRNPD lowered the steady-state levels of numerous target RNAs. Surprisingly, however, HNRNPD unexpectedly enhanced the steady-state levels of several target mRNAs encoding DNA-maintenance proteins. Accordingly, HNRNPD preserved genomic integrity in agreement with the AUF1-elicited prevention of premature cellular senescence. Currently, we are focusing on the KH-type splicing regulatory protein (KHSRP), an ARE-BP which is implicated in developmental processes and miRNA biogenesis, in addition to its proposed role in destabilizing target transcripts. We will integrate PAR-CLIP and RNAseq with CRISPR-Cas based genetic engineering to gain a complete overview of KHSRPs broad and vital role in post-transcriptional gene regulation.

Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2015
Total Cost
Indirect Cost
Name
Arthritis, Musculoskeletal, Skin Dis
Department
Type
DUNS #
City
State
Country
Zip Code
Min, Kyung-Won; Zealy, Richard W; Davila, Sylvia et al. (2018) Profiling of m6A RNA modifications identified an age-associated regulation of AGO2 mRNA stability. Aging Cell 17:e12753
Gogakos, Tasos; Brown, Miguel; Garzia, Aitor et al. (2017) Characterizing Expression and Processing of Precursor and Mature Human tRNAs by Hydro-tRNAseq and PAR-CLIP. Cell Rep 20:1463-1475
Yamaji, Masashi; Jishage, Miki; Meyer, Cindy et al. (2017) DND1 maintains germline stem cells via recruitment of the CCR4-NOT complex to target mRNAs. Nature 543:568-572
Benhalevy, Daniel; McFarland, Hannah L; Sarshad, Aishe A et al. (2017) PAR-CLIP and streamlined small RNA cDNA library preparation protocol for the identification of RNA binding protein target sites. Methods 118-119:41-49
Benhalevy, Daniel; Gupta, Sanjay K; Danan, Charles H et al. (2017) The Human CCHC-type Zinc Finger Nucleic Acid-Binding Protein Binds G-Rich Elements in Target mRNA Coding Sequences and Promotes Translation. Cell Rep 18:2979-2990
Arimbasseri, Aneeshkumar G; Iben, James; Wei, Fan-Yan et al. (2016) Evolving specificity of tRNA 3-methyl-cytidine-32 (m3C32) modification: a subset of tRNAsSer requires N6-isopentenylation of A37. RNA 22:1400-10
Danan, Charles; Manickavel, Sudhir; Hafner, Markus (2016) PAR-CLIP: A Method for Transcriptome-Wide Identification of RNA Binding Protein Interaction Sites. Methods Mol Biol 1358:153-73
Faraji, Farhoud; Hu, Ying; Yang, Howard H et al. (2016) Post-transcriptional Control of Tumor Cell Autonomous Metastatic Potential by CCR4-NOT Deadenylase CNOT7. PLoS Genet 12:e1005820
Løvendorf, Marianne B; Mitsui, Hiroshi; Zibert, John R et al. (2015) Laser capture microdissection followed by next-generation sequencing identifies disease-related microRNAs in psoriatic skin that reflect systemic microRNA changes in psoriasis. Exp Dermatol 24:187-93
Yoon, Je-Hyun; Jo, Myung Hyun; White, Elizabeth J F et al. (2015) AUF1 promotes let-7b loading on Argonaute 2. Genes Dev 29:1599-604

Showing the most recent 10 out of 14 publications