Regulation of RNA Polymerase II by 7sk
RNA Polymerase II (Pol II) generates a diverse set of coding and noncoding RNAs across the genome. The activity of Pol II transcription is highly regulated and occurs through number of diverse mechanisms. While traditional models focused on the initiation of transcription as the major point of regulation, more recent work has highlighted the importance of a subsequent step termed ‘pausing’. Pol II pausing is regulated by an inhibitory noncoding RNA named 7SK, which acts to directly inhibit the P-TEFb kinase. This mechanism was first described in 2001, and the functional importance of 7SK had been largely limited to the inhibition of the P-TEFb complex.
In an effort to discover novel activates for 7SK, we took an unbiased proteomics approach and identified the chromatin remodeler named BAF as a strong interacting complex with the 7SK RNA. Together with BAF, 7SK occupies mammalian enhancer elements to limit enhancer RNA transcription. Interestingly, upon depletion of 7SK, cells sustained increased DNA damage signaling and convergent transcription, especially at super enhancer regions (densely clustered enhancer elements). These data revealed a novel role for 7SK at enhancer elements, however the full extent of 7SK’s cellular role is likely still incomplete as it strongly partners with Pol II elongation complex members as well as the mRNA spliceosome. Future work will focus on elucidating the precise function of 7SK in different snRNA complexes across the mammalian nucleus.
Molecular functions of DEAD-box RNA helicases
Regulating RNA structure inside cells is an important layer in regulating RNA function. DEAD-box RNA helicases are a large group of ATP-dependent enzymes that can unwind RNA duplexes. With their ability to remodel RNA structure and act as RNA chaperones, DEAD-box proteins have been functionally implicated in nearly every step of RNA metabolism, from transcription and splicing, to translation and RNA degradation.
In collaboration with Joanna Wysocka's lab, we uncovered DDX21 as a central regulator of ribosomal biogenesis in both the nucleolus and nucleoplasm. DDX21 acts to enhance Pol I transcription and subsequently controls post-transcriptional methylation of the ribosomal RNA. Additionally, it positively regulates Pol II transcription at ribosomal protein genes via the 7SK snRNA as a P-TEFb release factor. What regulates the localization and activity of DDX21 remains an active area of research.
In collaboration with Dan Littman's lab, we identified DDX5 as a strongly interacting protein with the RORγt transcription factor, the factor that defines the differentiation signaling of Th17 CD4+ lymphocytes. DDX5 operates together with RORγt and a noncoding RNA named RMRP to promote the gene-expression network of RORγt. Surprisingly, mutations in the RMRP RNA, which are found in the cartilage-hair hypoplasia (CHH) disease, destabilized the DDX5-RORγt complex. This work establishes DDX5 as a player in T-cell differentiation and future efforts will be needed to molecularly define these RNA-protein interactions.
Exploration of additional DEAD-box proteins, and related RNA helicases, will provide insights into the full molecular mechanisms by which these proteins operate. Additionally, the integration of multiple helicase profiles will define networks of DEAD-box proteins operating together inside cells.
In vivo RNA secondary structure
RNA adopts complex physical conformations, importantly different structures can have similar energetics, providing modular and switch-like characteristics. Understanding the structural state of RNA is paramount for elucidating functional properties of a given transcript. We developed in vivo CLICK Selective 2’ Hydroxyl Acylation and Profiling Experiment (icSHAPE) which leverages a novel structure probe named NAI-N3. With this chemical and methodology, we can measure all four nucleobases at single-base resolution across the transcriptome of any organism.
While icSHAPE is a powerful tool to measure base-flexibility (and thus single stranded regions) in RNA molecules, it is “blind” to double-stranded regions. In an effort to address these drawbacks, we developed Psoralen Analysis of RNA Interactions by Sequencing or PARIS. Using PARIS, we are able to directly identify RNA duplexes across the transcriptome and these analyses revealed conserved RNA secondary structures, novel RNA-RNA interactions; including intra-mRNA interactions as well as inter-RNA contacts, and define the structure of the XIST RNA RepA region. Integrating icSHAPE, PARIS, and other structural techniques will provide a window into the complexities of the cellular transcriptome currently unappreciated.