Spliceosome activation: U4 is the path, stem I is the goal, and Prp8 is the keeper. Let's cheer for the ATPase Brr2!

In pre-mRNA splicing, introns are removed and exons are joined to generate mature mRNA. In the first of two transesterfication reactions required for splicing, the 2′ OH group of an adenosine within a conserved intronic branch site attacks the 5′ splice site (SS), generating a free 5′ exon and a lariat intermediate. In the second reaction, the newly liberated 3′ OH of the 5′ exon attacks the 3′ SS, generating mature mRNA and an excised intron lariat. These reactions are facilitated by five small nuclear RNAs (snRNAs) and 80 conserved proteins that together compose the spliceosome (Will and Luhrmann 2011). Throughout the splicing cycle, the spliceosome undergoes extraordinarily intricate and dynamic transformations involving exchanges of protein–protein, protein–RNA, and RNA–RNA interactions. These rearrangements are in many cases catalyzed by one of the eight spliceosomal members of the DExD/H-box ATPase family (Cordin et al. 2012) and must occur in a regulated and timely manner (Will and Luhrmann 2011). After de novo assembly on a pre-mRNA, spliceosomes are inactive and require a dramatic rearrangement of catalytic components for catalysis. The catalytic core of the spliceosome is composed of base-paired U2/U6 snRNAs (Madhani and Guthrie 1992). This interaction juxtaposes the reactants within the substrates, and the adjacent U6 intramolecular stem–loop (ISL) coordinates a divalent metal that functions at the catalytic stage (Yean et al. 2000). Importantly, neither base-paired U2/U6 nor the U6 ISL is preformed in spliceosomes. Instead U6 is base-paired with U4 within the U4/U6•U5 tri-snRNP in a mutually exclusive fashion. Thus, a key step in the activation of the spliceosome involves unwinding of U4/U6. This unwinding has been shown to depend on Brr2 and ATP in vivo and in vitro (Laggerbauer et al. 1998; Raghunathan and Guthrie 1998; Kim and Rossi 1999). This activity is compromised by mutations in human Brr2 that confer blindness in some cases of retinitis pigmentosa (Zhao et al. 2009). Because Brr2 is an integral factor of the U5 snRNP (Laggerbauer et al. 1996), the association of this snRNP with the di-snRNP U4/U6, a prerequisite for spliceosome assembly, brings Brr2 close to its substrate, U4/U6. Indeed, this U4/U6•U5 tri-snRNP can promote U4/U6 unwinding in an ATP-dependent manner in the absence of pre-mRNA (Raghunathan and Guthrie 1998). This premature activity is, however, antagonistic for spliceosome assembly, so U4/U6 unwinding must be timed appropriately (Small et al. 2006; Bellare et al. 2008; see below). While we know that Brr2 is required for U4/U6 unwinding, precisely how Brr2 promotes U4/U6 unwinding is unknown. The U4/U6 duplex consists of two adjacent stems (I and II) separated by an ISL in U4, named the 5′ SL. Based on structural homology with the DNA helicase Hel308, Brr2 is believed to function processively in a 3′-to-5′ direction (Buttner et al. 2007; Pena et al. 2009; Zhang et al. 2009; Santos et al. 2012). Thus, if it unwinds U4/U6 directly, Brr2 could initiate at the 3′ end of U6 and translocate along U6, unwinding stem II first and stem I second. Alternatively, Brr2 could initiate at the 3′ end of U4 and translocate along U4, unwinding stem I (Fig. 1). In this case, before Brr2 could unwind stem II, Brr2 would encounter the 5′ SL in U4, an element that is bound by proteins (Nottrott et al. 2002). In the November 1, 2012, issue of Genes & Development, two studies distinguished between these possibilities. Beggs and coworkers (Hahn et al. 2012), using in vivo cross-linking, and Luhrmann and coworkers (Mozaffari-Jovin et al. 2012), using in vitro functional assays, provided evidence that Brr2 translocates along U4 and initiates unwinding at U4/U6 stem I. This surprising result raises the question of how stem II is unwound and sets the stage for understanding how Brr2 is regulated. Figure 1. Prp8-regulated and Brr2-mediated unwinding of U4/U6. New results suggest a model for U4/U6 unwinding. (i) Hahn et al. (2012) showed that Brr2 (green oval) associates with the 3′ side of the 3′ SL of U4, a likely site of initial recruitment ...