Studies on RNA Regulation: From Enhancer RNAs to RBBP6 Isoform3

Abstract

Studies on RNA Regulation: From Enhancer RNAs to RBBP6 Isoform3 Yaqiong Chen This dissertation contains two separate yet interconnected pieces of work, which shed light on the complicated RNA regulatory mechanism. The first part, as the main focus of the thesis, characterizes a large pool of human polyadenylated enhancer RNA under deficient nuclear surveillance conditions, and investigates their metabolism mechanisms. The second part elucidates the dynamic localization mechanism of RBBP6 isoform3, which inhibits pre-mRNA 3’ processing by completing with RBBP6 isoform1. Despite being composed of approximately 3 billion base pairs, only 1 to 2% of the human genome codes for proteins. The non-coding DNA regions can however function as transcription units and generate non-coding RNAs such as enhancer-derived RNAs, or eRNAs, that play crucial roles in gene expression regulation, cell differentiation, development, and diseases. Previous studies have suggested that most eRNAs are transcribed by RNA polymerase II (RNAP II), but not polyadenylated. In Chapter 3, I identify a large fraction of polyadenylated enhancer RNAs under deficient nuclear surveillance conditions via genome-wide analyses, and explore their biogenesis and degradation mechanisms. I find that the Integrator complex plays an important role in polyadenylated eRNA biogenesis, and that their exosome-dependent degradation requires two cofactor complexes containing the RNA helicase Mtr4: the PAXT/PPC complex and the NEXT complex. Additionally, the canonical poly(A) polymerases PAP-α and PAP-γ play a major role in the 3’ end processing of pA+ eRNA. Finally, I show that under deficient nuclear surveillance conditions, pA+ eRNAs accumulate in the cytoplasm and associate with polysomes, suggesting that at least some might have translation potential. I also contributed to the discovery of two novel complexes both containing the RNA helicase Mtr4, which is a master player of the nuclear surveillance system. Mtr4 and ZFC3H1 form the PAXT/PPC complex, which facilitates the turnover of polyadenylated nuclear RNAs, including prematurely terminated RNAs (ptRNAs), upstream antisense RNAs (uaRNAs), and eRNAs (see the paper in Appendix II). Mtr4 also associates with NRDE2 to form a complex, functioning in the DNA damage response pathway (see the paper in Appendix III). These works provide additional insights into the complexity and significance of the RNA helicase Mtr4. In the second part of the thesis, presented in Chapter 4, I studied a polyadenylation factor known as Retinoblastoma-binding protein 6 (RBBP6). RBBP6 was initially identified as a large multidomain protein, interacting with tumor suppressors p53 and Rb. Later, its diverse roles were uncovered in cell cycle progression, apoptosis, nucleic acid metabolism, differentiation, and mRNA processing. RBBP6 protein has four isoforms, among which the shortest isoform, iso3, has only one domain: the DWNN (Domain With No Name) domain. The DWNN domain displays high similarities with ubiquitin, implying its function as a novel ubiquitin-like modifier. However, I show that the DWNN domain is actually not a ubiquitin-like modifier, but is itself ubiquitinated. Moreover, the monoubiquitylation of iso3 can facilitate its localization at chromatin. Additionally, I find that the C-terminal tail of iso3 also plays a role in iso3 chromatin localization, presumably by interacting with other factors of the polyadenylation machinery. Pulldown experiments of iso3 followed by mass spectrometry identified Importin7 as an iso3-interacting factor that assists its cytoplasmic retention. Our results identified novel mechanisms for the dynamic localization of RBBP6 iso3, which shed light on the role of iso3 in mRNA 3’ processing and disease.