The genomes of hyperthermophilic Archaea encode dozens of methylation guide, C͞D box small RNAs that guide 2 -O-methylation of ribose to specific sites in rRNA and various tRNAs. The genes encoding the Sulfolobus homologues of eukaryotic proteins that are known to be present in C͞D box small nucleolar ribonucleoprotein (snoRNP) complexes were cloned, and the proteins (aFIB, aNOP56, and aL7a) were expressed and purified. The purified proteins along with an in vitro transcript of the Sulfolobus sR1 small RNA were reconstituted in vitro, into an RNP complex. The order of assembly of the three proteins onto the RNA was aL7a, aNOP56, and aFIB. The complex was active in targeting S-adenosyl methionine (SAM)-dependent, site-specific 2 -O-methylation of ribose to a short fragment of ribosomal RNA (rRNA) that was complementary to the D box guide region of the sR1 small RNA. The presence of aFIB was essential for methylation; mutant proteins having amino acid replacements in the SAM-binding motif of aFIB were able to assemble into an RNP complex, but the resulting complexes were defective in methylation activity. These experiments define the minimal number of components and the conditions required to achieve in vitro RNA guide-directed 2 -O-methylation of ribose in a target RNA.T he eukaryotic nucleolus is a highly specialized organelle where rRNA is transcribed, processed, folded, and assembled along with ribosomal proteins into small and large ribosomal subunits (1-5). During this process, up to a hundred or more nucleotide modifications are introduced into the ribosomal RNA (rRNA) by two distinct families of small nucleolar ribonucleoprotein (snoRNP) complexes. The snoRNAs in these RNP complexes contain short antisense guide elements that are used to target modifications to specific locations within the rRNAs. One guide family, the C͞D box snoRNPs, targets site-specific 2Ј-O-methylation of ribose (6-9), and the other guide family, the H͞ACA snoRNPs, targets site-specific conversion of uridine to pseudouridine (10).The C͞D box snoRNAs are characterized by a bipartite structure with conserved C box (RUGAUGA) and D box (CUGA) motifs near their respective 5Ј and 3Ј ends and related CЈ (UGAUGA) and DЈ (CUGA) motifs near the center of the molecule. The antisense elements are located upstream of the D or DЈ motifs and are generally 10 or more nucleotides (nt) in length. Methylation is directed to the rRNA nucleotide that participates in a Watson-Crick base pair five nucleotides upstream from the start of the D or DЈ box; this is the N plus five rule (10-12). Although the general mechanism used by these RNP complexes in mediating modification has been deduced from in vivo biochemical and genetic observations, isolation and characterization of the structure and the in vitro activity of these guide complexes have not been described.The human C͞D box snoRNAs associate with several essential proteins, including fibrillarin, NOP56, and NOP58 (paralogous proteins derived from a gene duplication event), and a 15.5-kDa protein (8,(12)...

Mammalian X chromosome inactivation is one of the most striking examples of epigenetic gene regulation. Early in development one of the pair of approximately 160-Mb X chromosomes is chosen to be silenced, and this silencing is then stably inherited through subsequent somatic cell divisions. Recent advances have revealed many of the chromatin changes that underlie this stable silencing of an entire chromosome. The key initiator of these changes is a functional RNA, XIST, which is transcribed from, and associates with, the inactive X chromosome, although the mechanism of association with the inactive X and recruitment of facultative heterochromatin remain to be elucidated. This review describes the unique evolutionary history and resulting genomic structure of the X chromosome as well as the current understanding of the factors and events involved in silencing an X chromosome in mammals.

SummaryIt has been known for nearly half a century that coding and non-coding RNAs (mRNA, and tRNAs and rRNAs respectively) play critical roles in the process of information transfer from DNA to protein. What is both surprising and exciting, are the discoveries in the last decade that cells, particularly eukaryotic cells, contain a plethora of non-coding RNAs and that these RNAs can either possess catalytic activity or can function as integral components of dynamic ribonucleoprotein machines. These machines appear to mediate diverse, complex and essential processes such as intron excision, RNA modification and editing, protein targeting, DNA packaging, etc. Archaea have been shown to possess RNP complexes; some of these are authentic homologues of the eukaryotic complexes that function as machines in the processing, modification and assembly of rRNA into ribosomal subunits. Deciphering how these RNA-containing machines function will require a dissection and analysis of the component parts, an understanding of how the parts fit together and an ability to reassemble the parts into complexes that can function in vitro . This article summarizes our current knowledge about small-non-coding RNAs in Archaea, their roles in ribosome biogenesis and their relationships to the complexes that have been identified in eukaryotic cells.

Gas vesicles are intracellular, microbial flotation devices that consist of mainly one protein, GvpA. The formation of halobacterial gas vesicles occurs along a complex pathway involving 14 different gvp genes that are clustered in a genomic region termed the "vac region". Various vac regions found in Halobacterium salinarum (p-vac and c-vac), Haloferax mediterranei (mc-vac), and Natronobacterium vacuolatum (nv-vac) have been investigated. Except for the latter vac region, the arrangement of the gvp genes is identical. Single gvp genes have been mutated to study the effect on gas vesicle synthesis in transformants and to determine their possible function. Each vac region exhibits a characteristic transcription pattern, and regulatory steps have been observed at the DNA, RNA, and protein level, indicating a complex regulatory network acting during gas vesicle gene expression.

SummaryArchaea use ribonucleoprotein (RNP) machines similar to those found in the eukaryotic nucleolus to methylate ribose residues in nascent ribosomal RNA. The archaeal complex required for this 2 ¢ ¢ ¢ ¢ -O-ribosemethylation consists of the C/D box sRNA guide and three proteins, the core RNA-binding aL7a protein, the aNop56 protein and the methyltransferase aFib protein. These RNP machines were reconstituted in vitro from purified recombinant components, and shown to have methylation activity when provided with a simple target oligonucleotide, complementary to the sRNA guide sequence. To obtain a better understanding of the versatility and specificity of this reaction, the activity of reconstituted particles on more complex target substrates, including 5S RNA, tRNA Gln and 'double target' oligonucleotides that exhibit either direct or reverse complementarity to both the D ¢ ¢ ¢ ¢ and D box guides, has been examined. The natural 5S and tRNA Gln substrates were efficiently methylated in vitro , as long as the complementarity between guide and target was about 10 base pairs in length, and lacked mismatches. Maximal activity of double guide sRNAs required that both methylation sites be present in cis on the target RNA.