Structure and mechanism of a molecular rheostat, an RNA thermometer that modulates immune evasion by Neisseria meningitidis.

Neisseria meningitidis causes bacterial meningitis and septicemia. It evades the host complement system by upregulating expression of immune evasion factors in response to changes in temperature. RNA thermometers within mRNAs control expression of bacterial immune evasion factors, including CssA, in the 5'-untranslated region of the operon for capsule biosynthesis. We dissect the molecular mechanisms of thermoregulation and report the structure of the CssA thermometer. We show that the RNA thermometer acts as a rheostat, whose stability is optimized to respond in a small temperature range around 37°C as occur within the upper airways during infection. Small increases in temperature gradually open up the structure to allow progressively increased access to the ribosome binding site. Even small changes in stability induced by mutations of imperfect base pairs, as in naturally occurring polymorphisms, shift the thermometer response outside of the desired temperature range, suggesting that its activity could be modulated by pharmacological intervention.

The box H/ACA snoRNP assembly factor Shq1p is a chaperone protein homologous to Hsp90 cochaperones that binds to the Cbf5p enzyme.

Box H/ACA small nucleolar (sno) ribonucleoproteins (RNPs) are responsible for the formation of pseudouridine in a variety of RNAs and are essential for ribosome biogenesis, modification of spliceosomal RNAs, and telomerase stability. A mature snoRNP has been reconstituted in vitro and is composed of a single RNA and four proteins. However, snoRNP biogenesis in vivo requires multiple factors to coordinate a complex and poorly understood assembly and maturation process. Among the factors required for snoRNP biogenesis in yeast is Shq1p, an essential protein necessary for stable expression of box H/ACA snoRNAs. We have found that Shq1p consists of two independent domains that contain casein kinase 1 phosphorylation sites. We also demonstrate that Shq1p binds the pseudourydilating enzyme Cbf5p through the C-terminal domain, in synergy with the N-terminal domain. The NMR solution structure of the N-terminal domain has striking homology to the 'Chord and Sgt1' domain of known Hsp90 cochaperones, yet Shq1p does not interact with the yeast Hsp90 homologue in vitro. Surprisingly, Shq1p has stand-alone chaperone activity in vitro. This activity is harbored by the C-terminal domain, but it is increased by the presence of the N-terminal domain. These results provide the first evidence of a specific biochemical activity for Shq1p and a direct link to the H/ACA snoRNP.

How arginine-rich domains coordinate mRNA maturation events.

Maturation of RNA is highly regulated from transcription to post-transcriptional processing and localization to specific cellular compartments. The complexes that contribute to these events are in many cases well understood; however, many of the protein factors that coordinate and regulate these RNA processing events remain poorly characterized. Among them are arginine-rich domains, most commonly sequences rich in arginine/serine (RS domains) or arginine/glycine/glycine (RGG boxes), that often appear among factors and complexes involved in RNA processing. They are emerging as key yet poorly understood players in the assembly and coupling of RNA processing events.

The box H/ACA RNP assembly factor Naf1p contains a domain homologous to Gar1p mediating its interaction with Cbf5p.

Naf1 is an essential protein involved in the maturation of box H/ACA ribonucleoproteins, a group of particles required for ribosome biogenesis, modification of spliceosomal small nuclear RNAs and telomere synthesis. Naf1 participates in the assembly of the RNP at transcription sites and in the nuclear trafficking of the complex. The crystal structure of a domain of yeast Naf1p, Naf1Delta1p, reveals a striking structural homology with the core domain of archaeal Gar1, an essential protein component of the mature RNP; it suggests that Naf1p and Gar1p have a common binding site on the enzymatic protein component of the particle, Cbf5p. We propose that Naf1p is a competitive binder for Cbf5p, which is replaced by Gar1p during maturation of the H/ACA particle. The exchange of Naf1p by Gar1p might be prompted by external factors that alter the oligomerisation state of Naf1p and Gar1p. The structural homology with Gar1 suggests that the function of Naf1 involves preventing non-cognate RNAs from being loaded during transport of the particle by inducing a non-productive conformation of Cbf5.

A new alpha-helical extension promotes RNA binding by the dsRBD of Rnt1p RNAse III.

Rnt1 endoribonuclease, the yeast homolog of RNAse III, plays an important role in the maturation of a diverse set of RNAs. The enzymatic activity requires a conserved catalytic domain, while RNA binding requires the double-stranded RNA-binding domain (dsRBD) at the C-terminus of the protein. While bacterial RNAse III enzymes cleave double-stranded RNA, Rnt1p specifically cleaves RNAs that possess short irregular stem-loops containing 12-14 base pairs interrupted by internal loops and bulges and capped by conserved AGNN tetraloops. Consistent with this substrate specificity, the isolated Rnt1p dsRBD and the 30-40 amino acids that follow bind to AGNN-containing stem-loops preferentially in vitro. In order to understand how Rnt1p recognizes its cognate processing sites, we have defined its minimal RNA-binding domain and determined its structure by solution NMR spectroscopy and X-ray crystallography. We observe a new carboxy-terminal helix following a canonical dsRBD structure. Removal of this helix reduces binding to Rnt1p substrates. The results suggest that this helix allows the Rnt1p dsRBD to bind to short RNA stem-loops by modulating the conformation of helix alpha1, a key RNA-recognition element of the dsRBD.