Gene expression involves delicate regulations by RNAs and RNA-binding proteins (RBP). The overarching goal of our research is to understand how non-coding RNAs and RBPs contribute to gene regulation in human health and disease.
TSS-miRNA biogenesis
MicroRNAs (miRNAs) have been shown to affect diverse cellular pathways critical to human development and disease, underscoring the need to elucidate the mechanism by which their levels are regulated. Canonically, miRNAs are produced from primary transcripts that are cleaved by the nuclear Microprocessor complex, with the resulting precursor (pre-)miRNA hairpins exported from the nucleus by Exportin-5, and further processed by cytoplasmic Dicer. In 2018, we discovered an alternative biogenesis pathway that produces 7-methylguanosine-capped pre-miRNAs with 5´ extensions. miRNAs produced from this pathway were named transcription start site (TSS-) miRNAs. Our results expanded an unusual pathway that is distinct from canonical miRNA biogenesis in pre-miRNA synthesis, nuclear-cytoplasmic transport, Dicer processing and guide strand selection. It therefore impacts a broad spectrum of biological research areas from RNA pol II transcription, RNA processing and export, to studies of miRNA function and RNAi vector development.
tss-MIrna function
Given that TSS-miRNA are independent of Drosha-processing, in cancer cells with low Drosha expression, their levels are elevated compared to most miRNAs. To probe the function of TSS-miRNAs, we adopted an AGO-CLASH (cross linking and sequencing of hybrids) protocol. CLASH is a modified CLIP-seq (crosslinking and immunoprecipitation followed by RNA sequencing) method that physically connects AGO-bound miRNA and target mRNA, allowing for high-confidence identification of the miRNA targetome. By performing CLASH in colorectal cancer HCT116 cells lacking Drosha, we identified the targetome of TSS-miRNAs. We also made the novel discovery that the most abundant TSS-miRNA miR-320a down-regulates expression of the endoplasmic reticulum chaperone CALNEXIN and activates expression of the cell stress-inducible transcription factor ATF4. Therefore,miR-320a activates both the unfolded protein response and the downstream integrated stress response, pathways that contribute to oncogenesis
Target directed miRNA degradation
Starting in 2020, we investigated a mechanism called target RNA-directed miRNA degradation (TDMD), in which miRNA levels are controlled by the targets that form extensive base-pairing interaction with specific miRNAs. First discovered with viral and artificial transcripts, TDMD has only been demonstrated for three cellular RNA transcripts before 2021. With the establishment of CLASH, we pursued the new research direction of TDMD. The premises for transcriptome-wide identification of TDMD triggers in CLASH data is that target RNA and miRNAs are physically ligated, and therefore we can capture miRNA 3′ end A/U extensions, which often occurs during TDMD . From CLASH data sets obtained from six human and mouse cell lines, we predicted eighteen high-confidence TDMD triggers and experimentally validated eight. Therefore,we have substantially expanded the inventory of endogenous TDMD transcripts. Among these was BCL2L11, which encodes the BIM protein that can induce apoptosis. By degrading anti-apoptotic miR-221 and miR-222, the BCL2L11 TDMD trigger could enhance BIM-induced apoptosis. Our novel discovery supports an exciting new gene-regulatory model by which TDMD trigger sequences within mRNAs cooperate with the encoded proteins to control critical cellular functions. The next key step would be a full functional understanding and capturing the potential therapeutic value of bona fide TDMD triggers.
m6A modification of 7SK snRNA
In 2023, we discovered that 7SK small nuclear RNA contains high levels of N6-methyladenosine (m6A) in non-small cell lung cancer (NSCLC) cells and that m6A-7SK is essential in Pol II transcriptional control. m6A writer, METTL3, and the m6A eraser, ALKBH5, dynamically regulate 7SK at eight different sites. Importantly, specific removal of m6A on 7SK using a dCasRx-ALKBH5 system dampens global Pol II transcription and inhibits NSCLC cell growth. Mechanistically, removal of m6A modification induces 7SK conformational change and sequesters P-TEFb, leading to significant Pol II pausing and reduced transcriptional output. Our results reveal a new layer of transcriptional regulation by m6A modification on a non-coding RNA. More importantly, the specific reduction of m6A-7SK inhibits NSCLC cell growth. Our discovery impacts a broad spectrum of biological research areas, from Pol II transcription, m6A RNA modification, RNA structure, RNA-protein interaction, to studies of NSCLC biology and therapeutic development.
