Research

The Small Nucleolar RNAs Play Essential Roles in Ribosome Biogenesis

    The small nucleolar RNAs (snoRNAs) play crucial roles in ribosome biosynthesis. Using complementary sequences, the snoRNAs base pair with rRNA to guide the folding, site-specific nucleotide modification, and cleavage of precursor ribosomal RNA. Based upon conserved snoRNA sequence elements, this large population of guide RNAs has been divided into two major families designated the box C/D and the box H/ACA snoRNAs. The primary function of the box C/D snoRNAs is to guide 2'-O-methylation of targeted rRNA nucleotides while the box H/ACA snoRNAs direct the conversion of specific uridine residues to pseudouridine.

The Box C/D Core Motif and Intronic snoRNA Processing

    Our laboratory is investigating the biogenesis, structure, and function of the box C/D RNP nucleotide modification complexes using U14 as a model system for the eukaryotic snoRNPs. Early work from our laboratory reported the first intronic snoRNA and we subsequently demonstrated a novel pre-mRNA intron processing pathway for snoRNA biosynthesis. Our work then defined the terminal box C/D core motif, a folded RNA element essential for snoRNA processing, snoRNP assembly, and snoRNP transport to the nucleolus.

The box C/D core motif is folded into a Kink- or K-turn

Box C/D snoRNA Core and Accessory Proteins and snoRNP Assembly

    Work in our group has helped to define those nuclear proteins that bind the box C/D snoRNA and assemble the snoRNP complex. Four box C/D snoRNP core proteins (15.5kD, Nop56, Nop58, and fibrillarin) are tightly bound to the box C/D core motif and are essential for intronic snoRNA processing and accumulation of the mature box C/D snoRNA. These four core proteins are also important for the nucleotide modification reaction with fibrillarin possessing the 2'-O-methylase activity. Our investigations have also defined two snoRNP "accessory" proteins which are essential for snoRNA biogenesis. These proteins are transiently associated with the snoRNP complex in the nucleoplasm and are essential for snoRNP biogenesis. Most recently, we have been investigating the archaeal box C/D sRNP complex. The archaeal box C/D RNP utilizes three core proteins which are highly homologous to the eukaryotic core proteins for sRNP assembly and sRNA-guided 2'-O-methylation of targeted rRNA nucleotides.

Ongoing Investigations

The Role of Accessory Proteins p50 and p55 in snoRNP Biogenesis

    We are presently investigating the role of accessory proteins p50 and p55 in snoRNP biogenesis. Our working hypothesis is that these accessory proteins are required for snoRNP assembly and/or transport of the assembled snoRNP complex from the nucleoplasm into the nucleolus. Nuclear microinjection experiments using the Xenopus oocyte are determining those steps in the snoRNP biogenesis pathway that are dependent upon accessory proteins p50 and p55. In addition, in vitro RNP assembly experiments are determining those accessory protein:core protein interactions that are essential for p50 and p55 function in snoRNP biogenesis.

In Vitro Assembly of Eukaryotic snoRNP and Archaeal sRNP Complexes: Dissection of Box C/D RNP Structure and Methylation Function

    We are also investigating the structure and function of the box C/D RNP core complex. For these studies, we are using both the eukaryotic U14 snoRNP and archaeal sR8 sRNP complexes as model systems for investigation. We have cloned and expressed in bacterial cells all of the eukaryotic and archaeal box C/D RNP core proteins. We are now using these recombinant proteins to assemble in vitro both eukaryotic box C/D snoRNPs and archaeal box C/D sRNPs. Recent work with the sR8 sRNP of Methanoccocus jannaschii has assembled an enzymatically active archaeal box C/D RNP complex. This in vitro assembled sRNP guides methylation from both the terminal box C/D core RNP and its internal C'/D' RNP. Efficient methylation requires that both complexes be juxtaposed in the same sRNP indicating essential crosstalk interactions between the two complexes for maximal 2'-O-methylation activity. Additional experiments have revealed an evolution in the RNA-binding capabilities of the archaeal and eukaryotic RNP core proteins thus explaining the unique structural differences observed between the eukaryotic box C/D snoRNP and archaeal box C/D sRNP. Future experiments will exploit both the archaeal and eukaryotic in vitro assembly systems to reveal the architecture of this evolutionarily ancient RNA:protein enzyme and dissect the mechanisms of RNA-guided nucleotide modification that are critical for de novo ribosome biosynthesis.

Ordered Assembly of the Box C/D RNP Complex Electrophoretic mobility-shift analysis has been used to study box C/D RNP assembly. Binding of the archaeal core proteins is ordered with L7 binding first followed by Nop56/58 and fibrillarin.

In vitro Reconstituted Box C/D RNPs Direct Site-Specific 2'-O-Methylation of Targeted Nucleotides Enzyme activity of the reconstituted archael box C/D RNP complex was carried out by measuring the incorporation of 3H-methyl groups (donated from S-adenosyl methionine) into target RNAs. The two RNA targets are complementary to the box C/D RNA guide regions upstream of box D (D target) and box D' (D' target). Target RNAs already possessing a methyl group at the designate nucleotide (D-CH3 and D'-CH3) were used as controls. Over time, the in vitro assembled box C/D RNP directs 2'-O-methylation of both target RNA substrates.