Molecular Basis of Box C/D RNA-Protein Interactions

Abstract

Watkins et al., 1996; Samarsky et al., 1998). It is also clear that a fraction of snoRNAs, such as U3, U8, and U14, depend on the same sets of conserved box C/D sequences to carry out site-specific cleavage of ribo-somal RNAs (Baserga et al. Peculis and Steitz, 1994; Samarsky and Fournier, 1998; Watkins et al., 2002). Another type of snoRNP, the box H/ACA snoRNPs, catalyzes site-specific pseudouridyla-tion of rRNA by employing similar mechanisms of sub-Summary strate recognition and enzyme organization (Ofengand and Fournier, 1998; Venema and Tollervey, 1999; Kiss, We have determined and refined a crystal structure 2002; Rozhdestvensky et al., 2003). of the initial assembly complex of archaeal box C/D Earlier biochemical studies have shown that the con-sRNPs comprising the Archaeoglobus fulgidus (AF) served box C and D sequences are the sites of protein L7Ae protein and a box C/D RNA. The box C/D RNA assembly (Kuhn et al., 2002; Szewczak et al., 2002; Wat-forms a classical kink-turn (K-turn) structure and the kins et al., 2002). The first protein that interacts with the resulting protein-RNA complex serves as a distinct box C/D sequences is a member of an RNA binding platform for recruitment of the fibrillarin-Nop5p com-protein family. This protein was identified to be 15.5 kDa plex. The cocrystal structure confirms previously pro-(Watkins et al., 2000) in human and L7Ae in Archaea posed secondary structure of the box C/D RNA that (Kuhn et al., 2002). The initial interaction established includes a protruded U, a UU mismatch, and two between the RNA binding protein and the box C/D motif sheared tandem GA base pairs. Detailed structural is required for subsequent assembly of other snoRNP or comparisons of the AF L7Ae-box C/D RNA complex sRNP proteins, including fibrillarin, the eucaryal Nop56/ with previously determined crystal structures of L7Ae 58p, or the archaeal Nop5p proteins (Watkins et al., homologs in complex with functionally distinct K-turn 2002; Cahill et al., 2002; Rashid et al., 2003; Tran et al., RNAs revealed a set of remarkably conserved princi-2003). Although the Nop56/Nop5p proteins appear to ples in protein-RNA interactions. These analyses pro-interact directly with the conserved box C/D elements vide a structural basis for interpreting the functional upon binding of the 15.5 kDa/L7Ae protein (Cahill et al., roles of the box C/D sequences in directing specific 2002; Rashid et al., 2003; Watkins et al., 2002), fibrillarin assembly of box C/D sRNPs. is the subunit that catalyzes the actual methyl transfer reaction (Niewmierzycka and Clarke, 1999; Omer et al., Introduction 2000, 2002; Wang et al., 2000). Thus the recruitment of fibrillarin in the assembly of box C/D s(no)RNPs medi-Box C/D small nucleolar ribonucleoprotein particles ated by the 15.5 kDa/L7Ae protein suggests an impor-(snoRNPs) catalyze site-specific 2-O-methylation and tant functional role for 15.5 kDa/L7Ae in constructing the processing of ribosomal RNAs (rRNAs). These reactions RNA architecture around the active site before catal...

Kink turn sRNA folding upon L7Ae binding using molecular dynamics simulations

“…19 Three stable hydrogen bonds were found in our room temperature simulation for U18/K79, G19/N33, and G19/N34. This result is in good agreement with the structure analysis that U18 and G19 form important interactions with L7Ae.…”

Control of box C/D snoRNP assembly by N 6 ‐methylation of adenine

“…Note that when the ‐1n nucleotide is cytosine, this generates a GAC sequence on the non‐bulged strand (boxed) that is a potential target for N 6 ‐methylation of the conserved adenine at the 1n position. The two helical arms of the k‐turn are named C (canonical) and NC (non‐canonical) as indicated.Structure of a standard box C/D k‐turn, together with the chemical structure of the G1b·A1n and A2b·G2n trans sugar‐Hoogsteen G·A base pairs (PDB 1RLG 74). …”

“…Structure of a standard box C/D k‐turn, together with the chemical structure of the G1b·A1n and A2b·G2n trans sugar‐Hoogsteen G·A base pairs (PDB 1RLG 74). …”

Controllable molecular motors engineered from myosin and RNA

“…M6-RB (for Myosin VI - RNA-Binding) was generated by fusing myosin VI to the L7Ae kink-turn binding domain 22 . Following a design principle established in previous work on engineered myosin lever arms 6,7,9,19 , we made use of a helix-sharing junction in which the C-terminal helix of the truncated myosin VI lever arm was fused to the N-terminal helix of the L7Ae domain.…”

“…Following a design principle established in previous work on engineered myosin lever arms 6,7,9,19 , we made use of a helix-sharing junction in which the C-terminal helix of the truncated myosin VI lever arm was fused to the N-terminal helix of the L7Ae domain. Guided by crystal structures of L7Ae-RNA 22 and of myosin VI 23,24 , we aligned the terminal helices and optimized the phasing of the junction to orient a bound kink-turn motif as a structural foundation from which to build extended RNA lever arms. The interaction of L7Ae with kink-turn motifs has been previously exploited in nanotechnology and synthetic biology applications 25–27 , and yields stable complexes with reported K d values of ~1 nM and dissociation rates of ~2–7 × 10 −4 s −1 25,27,28 .…”