Academic journal article Genetics

Genome-Wide Discovery of DEAD-Box RNA Helicase Targets Reveals RNA Structural Remodeling in Transcription Termination

Academic journal article Genetics

Genome-Wide Discovery of DEAD-Box RNA Helicase Targets Reveals RNA Structural Remodeling in Transcription Termination

Article excerpt

RNA helicases are found in all kingdoms of life, playing central roles in all aspects of RNA metabolism (Bourgeois et al. 2016). Among them, DEAD-box proteins constitute the largest RNA helicase family. These enzymes have a conserved helicase core, which is responsible for ATP binding, hydrolysis, and RNA binding, and are characterized by the AspGlu-Ala-Asp (D-E-A-D) motif. Most steps in gene expression involve DEAD-box helicases, including transcription, translation and RNA decay (Linder and Jankowsky 2011). However, the detailed molecular actions of these helicases remain to be characterized.

Most DEAD-box helicases have an ATP-dependent RNAunwinding activity in vitro (Putnam and Jankowsky 2013). This activity catalyzes a wide variety of biochemically distinct actions including nonprocessive, RNA duplex unwinding (Rogers et al. 1999; Yang and Jankowsky 2006), RNA-protein complex (RNP) remodeling activity in vitro (Fairman et al. 2004; Tran et al. 2007), and ATP-dependent "clamping" of multiprotein complexes onto RNA (Ballut et al. 2005; Nielsen et al. 2008). Studies have also shown that DEAD-box helicases can act as chaperones to promote RNA folding both in vitro and in vivo (Yang and Jankowsky 2005; Tijerina et al. 2006; Liebeg et al. 2010; Potratz et al. 2011). For example, Mss116 in Saccharomyces cerevisiae assists the folding of functional group I and II introns by unwinding misfolded RNAs to allow exchange between kinetically trapped, nonfunctional structures and functional conformations (Liebeg et al. 2010; Potratz et al. 2011). Human DEAD-box helicases, including DDX5 and DDX17, have also been reported to unwind secondary RNA structures and thereby regulate alternative splicing (Kar et al. 2011; Dardenne et al. 2014). RNA remodeling activity also appears to be critical for translation, as a recent genome-wide study of the translation factor and DEAD-box helicase Ded1 showed that this enzyme resolves structures in the 5' ends of genes and controls translational start site choice in S. cerevisiae (Guenther et al. 2018). These examples implicate DEAD-box helicases as potential regulators of RNA metabolism and gene expression, yet we lack a thorough understanding of how RNA structure and RNP assembly affect basic molecular steps within these processes.

The basic steps in transcription include initiation, elongation, and termination. Termination by RNA polymerase II (RNAPII) is mediated mainly by two complexes in S. cerevisiae: the cleavage and polyadenylation factor (CPF) complex and the Nrd1-Nab3-Sen1 (NNS) complex. CPF-dependent 3' end processing is the primary mode of termination for messenger RNA (mRNA) genes, whereas the NNS complex, a trimeric assembly of RNA-binding proteins Nrd1 and Nab3 with the RNA-DNA helicase Sen1, promotes termination of short, noncoding RNAPII transcripts and some mRNAs (Rondón et al. 2009; Porrua and Libri 2015). The NNS complex has also been implicated in "failsafe" termination, whereby NNS target sites can rescue defective termination from an upstream CPF-dependent site to prevent aberrant gene expression (Rondón et al. 2009).

Previous results from our laboratory showed that the ortholog of DDX5 in S. cerevisiae, Dbp2 (Xing et al 2019), is required for efficient termination of RNAPII transcription, as loss of DBP2 results in accumulation of a 3' extended GAL10 mRNA and GAL10s long, noncoding RNA (Cloutier et al. 2012). Both Dbp2 and DDX5 exhibit highly efficient RNA duplex unwinding in vitro, consistent with a role in altering secondary structure (Ma et al. 2013; Xing et al. 2017). Furthermore, Dbp2 associates with actively transcribed chromatin in an RNA-dependent manner (Ma et al. 2016) and is required for pre-mRNA maturation and messenger RNP assembly, as evidenced by reduced binding of export factors Nab2, Yra1, and Mex67 in dbp2D cells (Ma et al. 2013). As efficient termination is necessary for proper assembly of mRNA export factors (Qu et al. 2009), these two steps are likely linked through a common, upstream biochemical step mediated by Dbp2. …

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