Gene Expression Regulation by PABPN1 Phosphorylation and Microexon Splicing

ABSTRACT

The processing of pre-mRNAs into mature mRNAs is a key regulatory step in eukaryotic gene expression programs. Before an mRNA can be exported to the cytoplasm, its 5′ end must be modified with a 7-methylguanosine cap structure, intervening non-coding sequence must be removed (spliced), and a poly(A) tail must be synthesized at its 3′ end. Each of these processes are dynamic and provide contextual regulation, tuning an mRNAs coding capacity, stability, and translation potential as needed. The Neugebauer Lab has developed highly specialized tools to dissect how RNAs are spliced, cleaved, and polyadenylated in numerous biological systems. In this study, I applied these tools and other biochemical methods to answer two outstanding questions related to how RNAs are processed: (1) How does the Nuclear Poly(A) Binding Protein, PABPN1, regulate poly(A) tailing and RNA turnover during mitosis in cycling human cells? and (2) How are small microexons defined and spliced into RNAs that are still undergoing synthesis? The eukaryotic cell cycle is orchestrated by waves of phosphorylation by cyclindependent kinases, which regulate numerous cellular processes required for cell division. The Neugebauer Lab previously discovered that human PABPN1 is phosphorylated by mitotic kinases at four specific sites during mitosis, highlighting a potential role for PABPN1 in regulating the mitotic transcriptome. PABPN1 helps maintain processivity of the canonical poly(A) polymerase (PAP) by oligomerizing on poly(A) tails and stabilizing the PAP-RNA interaction. PABPN1 is also involved in decay of a select class of nuclear RNAs. Given these two functions, it is surprising that poly(A) tails remain constant between mitosis and interphase, as nuclear envelope breakdown precedes mixing of the nuclear and cytoplasmic compartments. This implies tight regulation of the poly(A) tailing machinery during mitosis, possibly by PABPN1 phosphorylation. To understand the functional consequences of PABPN1 phosphorylation in cycling cells, I used a panel of stable cell lines capable of inducible over-expression of PABPN1 with point mutations at mitotic phosphorylation sites. Expression of phospho-inhibitory PABPN1 resulted in decreased cell proliferation, highlighting the importance of PABPN1 phosphorylation in cycling cells. Poly(A) tail length can drastically impact RNA stability and has emerged as an important mode of gene expression regulation in development. I first employed long-read sequencing to probe the effects of PABPN1 phospho-mutant expression on poly(A) tailing transcriptome-wide. I discovered that expression of phospho-inhibitory PABPN1 leads to widespread hyperadenylation of RNA, while phospho-mimetic PABPN1 has little effect on poly(A) tail lengths. In contrast, phospho-mimetic PABPN1 resulted in increased frequency of non-A nucleotide incorporation in tails, which can have additional impacts on RNA stability. To directly measure RNA turnover, TimeLapse-seq was employed following expression of PABPN1 phospho-mutants. Phospho-mimetic PABPN1 expression resulted in reduced stability for a subset of RNAs. However, little correlation was observed between changes in poly(A) tail length and changes in RNA turnover. Rather, PABPN1 phosphorylation appears to affect RNA stability by mechanisms other than poly(A) tail length regulation. The results of this study indicate that PABPN1 phosphorylation remodels poly(A) tails and increases mRNA turnover, supporting a model that enhanced transcriptome dynamics reset gene expression programs across the cell cycle. In a parallel study, I applied tools previously developed in the Neugebauer Lab to directly probe co-transcriptional splicing of alternatively spliced microexons. Alternative pre-mRNA splicing is a key regulatory step in mammalian gene expression and is at the heart of numerous developmental programs. The inclusion or exclusion of alternative exons can produce diverse transcripts from a single gene and provides a platform to regulate RNA stability and protein composition. Although delayed splicing has been reported for introns flanking many alternative exons, I wondered if alternative splice site selection can also take place rapidly and efficiently during synthesis as many constitutive splicing events do. Recent work has highlighted the importance of tissue-specific microexons, which are highly conserved 3-27 nucleotide-long cassette exons. To address whether microexons are defined and spliced co-transcriptionally, I isolated nascent RNA from Neuro-2a cells and performed short and long-read RNA sequencing, mapping polymerase II position and splicing status of single RNA molecules. In this study, I report that microexons that are dependent on the splicing factor SRRM4 are efficiently co-transcriptionally spliced in Neuro-2a cells, similar to longer constitutive exons. I then focused on the sequence elements that enable alternative cotranscriptional splicing of microexons. To more efficiently screen for cis-regulatory elements that impact microexon recognition, I generated several minigenes with variable sequences surrounding microexon splice sites. By substituting the endogenous weaker 5′ splice site with a strong one, the alternatively spliced microexon was constitutively spliced independent of any known microexon splicing factors, such as SRRM4. Rapid co- transcriptional definition thereby coordinates the co-transcriptional excision of introns upstream and downstream of microexons. Taken together, the work described in this thesis fills in substantial gaps in our understanding of cell-cycle dependent RNA stability and polyadenylation and identifies several molecular determinants for co-transcriptional microexon splicing.