Signature amino acids enable the archaeal L7Ae box C/D RNP core protein to recognize and bind the K-loop RNA motif

Abstract

The archaeal L7Ae and eukaryotic 15.5kD protein homologs are members of the L7Ae/15.5kD protein family that characteristically recognize K-turn motifs found in both archaeal and eukaryotic RNAs. In Archaea, the L7Ae protein uniquely binds the K-loop motif found in box C/D and H/ACA sRNAs, whereas the eukaryotic 15.5kD homolog is unable to recognize this variant K-turn RNA. Comparative sequence and structural analyses, coupled with amino acid replacement experiments, have demonstrated that five amino acids enable the archaeal L7Ae core protein to recognize and bind the K-loop motif. These signature residues are highly conserved in the archaeal L7Ae and eukaryotic 15.5kD homologs, but differ between the two domains of life. Interestingly, loss of K-loop binding by archaeal L7Ae does not disrupt C9/D9 RNP formation or RNA-guided nucleotide modification. L7Ae is still incorporated into the C9/D9 RNP despite its inability to bind the K-loop, thus indicating the importance of protein-protein interactions for RNP assembly and function. Finally, these five signature amino acids are distinct for each of the L7Ae/L30 family members, suggesting an evolutionary continuum of these RNA-binding proteins for recognition of the various K-turn motifs contained in their cognate RNAs.

YbxF and YlxQ are bacterial homologs of L7Ae and bind K-turns but not K-loops

“…This also shows that the shortened a4-b4 loop on YlxQ is not principally responsible for its reduced affinity toward K-turns. This result parallels a mutational study of L7Ae in which a conserved lysine (K26 in M. jannaschii L7Ae) in the a1-b1 loop, which does not interact directly with RNA, was found to modulate RNA affinity (Gagnon et al 2010). …”

“…Previously, it was hypothesized that the specific sequence of the a4-b4 loop (which differs between the archaeal and eukaryal family members) was responsible for the ability of L7Ae to bind K-loops (Hamma and Ferré-D'Amaré 2004). Gagnon et al (2010) found that substitution of the L7Ae a4-b4 loop with the corresponding loop of 15.5kD abrogated the ability of the archaeal protein to bind to K-loops, thus showing that these amino acids are necessary for K-loop binding by L7Ae. However, those investigators did not produce the converse mutant (15.5kD protein with the L7Ae a4-b4 loop), and thus it is unclear if this loop suffices for K-loop binding.…”

2'-O-methylation of the wobble residue of elongator pre-tRNAMetinHaloferax volcaniiis guided by a box C/D RNA containing unique features

“…25,28,29,31,47 Therefore, sR-tMet RNA was mixed with increasing concentrations of the L7Ae protein to determine if the box C'/D' motif lacking a K-turn or K-loop can also bind the protein. Gel-shift analyses of assembled RNPs suggest that at lower concentrations of protein, only one ribonucleoprotein (RNP) is formed (RNP1 in Fig.…”

“…Archaeal L7Ae protein can bind to both a K-turn and a K-loop in box C/D and box H/ACA RNAs. 10,16,23,[27][28][29][56][57][58] Titration of guide RNAs that contain both box C/D and C'/D' motifs with K-turn/K-loop, show formation of two distinct RNP complexes with increasing concentrations of L7Ae. 25,28,29,31,47 H. volcanii sR-tMet can also form two distinct RNP complexes with increasing concentrations of L7Ae (Fig.…”

“…11 A stretch of 10-21 nucleotides 5' of box D and sometimes box D' pair with their RNAs. 10,[26][27][28][29] The canonical K-turn consists of a 3-base bulge flanked on its 5' side by a normal base-paired canonical stem (C stem) and on its 3' side by a non-canonical stem (NC stem) containing tandem, sheared G•A base pairs. 27,30 All known modified versions of K-turns contain an asymmetric bulge between these two stems (www.dundee.ac.uk/biocentre/nasg/kturn/ index.php).…”