Date of Award
Doctor of Philosophy (PhD)
Molecular, Cellular, and Developmental Biology
Pervasive transcription is a hallmark of mammalian genomes. Although protein-coding genes span only a small fraction of the genome, more than two-thirds is transcribed, yielding thousands of noncoding transcripts whose expression exhibits a tight correlation with cell type, disease state, and other biological phenomena. A subset of these transcripts, termed long noncoding RNAs (lncRNAs) on account of their length (>200 nucleotides) and lack of apparent coding potential, have been shown to play functional roles in processes ranging from immune signaling to organogenesis. In contrast to trans-acting lncRNAs, which may operate in either the nucleus or cytoplasm, cis-acting lncRNAs remain at their site of transcription and regulate the expression of nearby protein-coding genes. These lncRNAs have been proposed to act through three main mechanisms: (1) the RNA molecule may interact with protein factors to enact transcriptional activation or repression; (2) the act of lncRNA transcription may increase the local concentration of RNA polymerase II or chromatin-modifying factors; or (3) DNA elements within a lncRNA locus may directly regulate the expression of both the lncRNA and its neighboring gene. Deconvolving these interlinked mechanisms has proven challenging and necessitates the development and implementation of new experimental techniques. In this work, we used a suite of independent molecular and genetic approaches, including a novel ribozyme-based tool for targeted transcript degradation, to expand our understanding of the molecular “logic” through which cis-acting lncRNAs enact gene regulation. We focused on the p53-inducible lncRNA LincRNA-p21, which acts in cis to reinforce the expression of the nearby protein-coding gene and key p53 target p21/Cdkn1a. To identify the functional element of cis-regulation at this locus, we generated four mouse strains harboring complementary LincRNA-p21 loss-of-function mutations that allowed us to separately examine the importance of the LincRNA-p21 transcriptional process and the underlying DNA sequence. This parallel genetic approach demonstrated that full-length LincRNA-p21 transcription, processing, and accumulation are dispensable for cis-regulation and revealed a requirement for a conserved sequence element within exon 1. Further experiments with molecular tools suggested that active transcription through this conserved region promotes p21 expression, implicating a dual role for the transcriptional process and sequence elements within the locus. This comprehensive functional dissection of a single lncRNA locus attests to the regulatory potential of lncRNA loci and further reveals the biological significance of pervasive genomic transcription.
Winkler, Lauren Nicole, "Elucidating the Molecular Mechanism of Cis-Regulation by the Long Noncoding RNA LincRNA-p21" (2021). Yale Graduate School of Arts and Sciences Dissertations. 440.