A dimeric structure for archaeal box C/D small ribonucleoproteins.
September 14, 2009
Bleichert F, Gagnon KT, Brown BA 2nd, Maxwell ES, Leschziner AE, Unger VM, Baserga SJ.
Science. 2009 Sep 11;325(5946):1384-7.Methylation of ribosomal RNA (rRNA) is required for optimal protein synthesis. Multiple 2'-O-ribose methylations are carried out by box C/D guide ribonucleoproteins [small ribonucleoproteins (sRNPs) and small nucleolar ribonucleoproteins (snoRNPs)], which are conserved from archaea to eukaryotes. Methylation is dictated by base pairing between the specific guide RNA component of the sRNP or snoRNP and the target rRNA. We determined the structure of a reconstituted and catalytically active box C/D sRNP from the archaeon Methanocaldococcus jannaschii by single-particle electron microscopy. We found that archaeal box C/D sRNPs unexpectedly formed a dimeric structure with an alternative organization of their RNA and protein components that challenges the conventional view of their architecture. Mutational analysis demonstrated that this di-sRNP structure was relevant for the enzymatic function of archaeal box C/D sRNPs.
A structural model for the HAT domain of Utp6 incorporating bioinformatics and genetics.
July 30, 2009
Champion EA, Kundrat L, Regan L, Baserga SJ.
Protein Eng Des Sel. 2009 Jul;22(7):431-9.The half-a-tetratricopeptide (HAT) repeat motif is of interest because it is found exclusively in proteins that are involved in RNA metabolism. Little is known about structure-function relationships in this class of repeat motif. Here, we present the results of a combined bioinformatics, modeling and mutagenesis study of the HAT domain of Utp6. We have derived a new HAT consensus, delineated its structure-defining residues and, by homology modeling, have placed these residues in a structural context. By considering only HAT motifs from Utp6 proteins, we identified residues that are shared by, and unique to, only this subset of HAT motifs, suggesting a key functional role. Employing both random and directed mutagenesis of the HAT domain in yeast Utp6, we have identified residues whose mutation results in loss of function. By examining these residues in the context of the homology model, we have delineated those that act by perturbing structure and those that more likely have a direct effect on function. Importantly, the residues we predict to have a direct effect on function map together on the tertiary structure, thus defining a potential functional interaction surface.
A direct interaction between the Utp6 HAT domain and a specific peptide in Utp21 is essential for efficient pre-rRNA processing.
September 08, 2008
Champion EA, Lane BH, Jackrel ME, Regan L, Baserga SJ.
Mol Cell Biol. 2008 Aug 25. [Epub ahead of print]The SSU processome is a ribosome biogenesis intermediate that assembles from its subcomplexes onto the pre-18S rRNA with yet unknown order and structure. Here, we investigate the architecture of the UtpB subcomplex of the SSU processome, focusing on the interaction between the HAT domain of Utp6 and a specific peptide in Utp21. We present a comprehensive map of the interactions within the UtpB subcomplex, and further show that the N-terminal domain of Utp6 interacts with Utp18, while the HAT domain interacts with Utp21. Using a panel of point and deletion mutants of Utp6, we show that an intact HAT domain is essential for efficient pre-rRNA processing and cell growth. Further investigation of the Utp6-Utp21 interaction using both genetic and biophysical methods shows that the HAT domain binds a specific peptide ligand in Utp21, the first example of a HAT domain peptide ligand, with a dissociation constant of 10 microM.
The long unwinding road of RNA helicases.
August 03, 2007
Bleichert F, Baserga SJ.
Mol Cell. 2007 Aug 3;27(3):339-52.RNA helicases comprise a large family of enzymes that are thought to utilize the energy of NTP binding and hydrolysis to remodel RNA or RNA-protein complexes, resulting in RNA duplex strand separation, displacement of proteins from RNA molecules, or both. These functions of RNA helicases are required for all aspects of cellular RNA metabolism, from bacteria to humans. We provide a brief overview of the functions of RNA helicases and highlight some of the recent key advances that have contributed to our current understanding of their biological function and mechanism of action.
Ribosome biogenesis is sensed at the start cell cycle checkpoint.
March 18, 2007
Bernstein KA, Bleichert F, Bean JM, Cross FR, Baserga SJ.
Mol Biol Cell. 2007 Mar;18(3):953-64. Epub 2006 Dec 27. In the yeast Saccharomyces cerevisiae it has long been thought that cells must reach a critical cell size, called the "setpoint," in order to allow the Start cell cycle transition. Recent evidence suggests that this setpoint is lowered when ribosome biogenesis is slowed. Here we present evidence that yeast can sense ribosome biogenesis independently of mature ribosome levels and protein synthetic capacity. Our results suggest that ribosome biogenesis directly promotes passage through Start through Whi5, the yeast functional equivalent to the human tumor suppressor Rb. When ribosome biogenesis is inhibited, a Whi5-dependent mechanism inhibits passage through Start before significant decreases in both the number of ribosomes and in overall translation capacity of the cell become evident. This delay at Start in response to decreases in ribosome biogenesis occurs independently of Cln3, the major known Whi5 antagonist. Thus ribosome biogenesis may be sensed at multiple steps in Start regulation. Ribosome biogenesis may thus both delay Start by increasing the cell size setpoint and independently may promote Start by inactivating Whi5.