Tamás Kiss

Small nucleolar RNAs: An abundant group of noncoding RNAs with diverse cellular functions

Small nucleolar RNAs represent an abundant, evolutionarily ancient group of noncoding RNAs which possess impressively diverse functions ranging from 2-O-methylation and pseudouridylation of various classes of RNAs, through nucleolytic processing of rRNAs to the synthesis of telomeric DNA.

Site-Specific Ribose Methylation of Preribosomal RNA: A Novel Function for Small Nucleolar RNAs

Eukaryotic cells contain many fibrillarin-associated small nucleolar RNAs (snoRNAs) that possess long complementarities to mature rRNAs. Characterization of 21 novel antisense snoRNAs from human cells followed by genetic depletion and reconstitution studies on yeast U24 snoRNA provides evidence that this class of snoRNAs is required for site-specific 2-O-methylation of preribosomal RNA (pre-rRNA). Antisense sno-RNAs function through direct base-pairing interactions with pre-rRNA. The antisense element, together with the D or D box of the snoRNA, provide the information necessary to select the target nucleotide for the methyltransfer reaction. The conclusion that sno-RNAs function in covalent modification of the sugar moieties of ribonucleotides demonstrates that eukaryotic small nuclear RNAs have a more versatile cellular function than earlier anticipated.

7SK small nuclear RNA binds to and inhibits the activity of CDK9/cyclin T complexes

The transcription of eukaryotic protein-coding genes involves complex regulation of RNA polymerase (Pol) II activity in response to physiological conditions and developmental cues. One element of this regulation involves phosphorylation of the carboxy-terminal domain (CTD) of the largest polymerase subunit by a transcription elongation factor, P-TEFb, which comprises the kinase CDK9 and cyclin T1 or T2 (ref. 1). Here we report that in human HeLa cells more than half of the P-TEFb is sequestered in larger complexes that also contain 7SK RNA, an abundant, small nuclear RNA (snRNA) of hitherto unknown function. P-TEFb and 7SK associate in a specific and reversible manner. In contrast to the smaller P-TEFb complexes, which have a high kinase activity, the larger 7SK/P-TEFb complexes show very weak kinase activity. Inhibition of cellular transcription by chemical agents or ultraviolet irradiation trigger the complete disruption of the P-TEFb/7SK complex, and enhance CDK9 activity. The transcription-dependent interaction of P-TEFb with 7SK may therefore contribute to an important feedback loop modulating the activity of RNA Pol II.

Site-Specific Pseudouridine Formation in Preribosomal RNA Is Guided by Small Nucleolar RNAs

During the nucleolar maturation of eukaryotic ribosomal RNAs, many selected uridines are converted into pseudouridine by a thus far undefined mechanism. The nucleolus contains a large number of small RNAs (snoRNAs) that share two conserved sequence elements, box H and ACA. In this study, we demonstrate that site-specific pseudouridylation of rRNAs relies on short ribosomal signal sequences that are complementary to sequences in box H/ACA snoRNAs. Genetic depletion and reconstitution studies on yeast snR5 and snR36 snoRNAs demonstrate that box H/ACA snoRNAs function as guide RNAs in rRNA pseudouridylation. These results define a novel function for snoRNAs and further reinforce the idea that base pairing is the most common way to obtain specific substrate-enzyme interactions during rRNA maturation.

Cajal body-specific small nuclear RNAs: a novel class of 2′-O-methylation and pseudouridylation guide RNAs

Cajal (coiled) bodies are conserved subnuclear organelles that are present in the nucleoplasm of both animal and plant cells. Although Cajal bodies were first described nearly 100 years ago, their function has remained largely speculative. Here, we describe a novel class of human small nuclear RNAs that localize specifically to Cajal bodies. The small Cajal body‐ specific RNAs (scaRNAs) are predicted or have already been demonstrated to function as guide RNAs in site‐specific synthesis of 2′‐O‐ribose‐methylated nucleotides and pseudouridines in the RNA polymerase II‐transcribed U1, U2, U4 and U5 spliceosomal small nuclear RNAs (snRNAs). Our results provide strong support for the idea that the Cajal body, this mysterious nuclear organelle, provides the cellular locale for post‐transcriptional modification of spliceosomal snRNAs.

Function and synthesis of small nucleolar RNAs

Eukaryotic cells contain an extraordinarily complex population of small nucleolar RNAs (snoRNAs). During its brief lifetime, each human pre-rRNA molecule will transiently associate with approximately 150 different snoRNA species. In the past year our understanding of snoRNAs has been clarified by the recognition that the snoRNA population can be divided into a small number of groups which are structurally and functionally distinct. The two largest groups of snoRNAs direct the site-specific modification of the pre-rRNA at positions of 2-O-methylation and pseudouridine formatio. Other groups of snoRNAs function in pre-rRNA cleavage and in the formation of the correct structure of the pre-rRNA.

Modification of Sm small nuclear RNAs occurs in the nucleoplasmic Cajal body following import from the cytoplasm

Biogenesis of functional spliceosomal small nuclear RNAs (snRNAs) includes the post‐transcriptional covalent modification of numerous internal nucleotides. We have recently demonstrated that synthesis of 2′‐O‐methylated nucleotides and pseudouridines in the RNA polymerase II‐synthesized Sm snRNAs is directed by sequence‐specific guide RNAs. Here, we provide evidence supporting the notion that modification of Sm snRNAs occurs in nucleoplasmic Cajal bodies (CBs), where modification guide RNAs accumulate. We show that short fragments of Sm snRNAs are correctly and efficiently modified when targeted to CBs, but not when these same fragments are targeted to the nucleolus. We also demonstrate that internal modification of the U2 snRNA occurs exclusively after nuclear import of the newly assembled Sm snRNP from the cytoplasm. Finally, we show that p80 coilin, the CB marker protein, is not required for snRNA modification. In coilin knockout cells, Sm snRNAs and their modification guide RNAs colocalize in residual CBs, which do not stockpile fibrillarin and fail to recruit the U3 small nucleolar RNA.

Sequence and structural elements of methylation guide snoRNAs essential for site-specific ribose methylation of pre-rRNA

Site‐specific 2′‐O‐ribose methylation of eukaryotic rRNAs is guided by small nucleolar RNAs (snoRNAs). The methylation guide snoRNAs carry long perfect complementaries to rRNAs. These antisense elements are located either in the 5′ half or in the 3′ end region of the snoRNA, and are followed by the conserved D′ or D box motifs, respectively. An uninterrupted helix formed between the rRNA and the antisense element of the snoRNA, in conjunction with the adjacent D′ or D box, constitute the recognition signal for the putative methyltransferase. Here, we have identified an additional essential box element common to methylation guide snoRNAs, termed the C′ box. We show that the C′ box functions in concert with the D′ box and plays a crucial role in the methyltransfer reaction directed by the upstream antisense element and the D′ box. We also show that an internal fragment of U24 methylation guide snoRNA, encompassing the upstream antisense element and the D′ and C′ box motifs, can support the site‐specific methylation of rRNA. This strongly suggests that the C box of methylation guide snoRNAs plays an essential role in the methyltransfer reaction guided by the 3′‐terminal antisense element and the D box of the snoRNA.

The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery

RNA-binding proteins of the L7Ae family are at the heart of many essential ribonucleoproteins (RNPs), including box C/D and H/ACA small nucleolar RNPs, U4 small nuclear RNP, telomerase, and messenger RNPs coding for selenoproteins. In this study, we show that Nufip and its yeast homologue Rsa1 are key components of the machinery that assembles these RNPs. We observed that Rsa1 and Nufip bind several L7Ae proteins and tether them to other core proteins in the immature particles. Surprisingly, Rsa1 and Nufip also link assembling RNPs with the AAA + adenosine triphosphatases hRvb1 and hRvb2 and with the Hsp90 chaperone through two conserved adaptors, Tah1/hSpagh and Pih1. Inhibition of Hsp90 in human cells prevents the accumulation of U3, U4, and telomerase RNAs and decreases the levels of newly synthesized hNop58, hNHP2, 15.5K, and SBP2. Thus, Hsp90 may control the folding of these proteins during the formation of new RNPs. This suggests that Hsp90 functions as a master regulator of cell proliferation by allowing simultaneous control of cell signaling and cell growth.

Box H/ACA Small Ribonucleoproteins

Box H/ACA RNAs represent an abundant, evolutionarily conserved class of small noncoding RNAs. All H/ACA RNAs associate with a common set of proteins, and they function as ribonucleoprotein (RNP) enzymes mainly in the site-specific pseudouridylation of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). Some H/ACA RNPs function in the nucleolytic processing of precursor rRNA (pre-rRNA) and synthesis of telomeric DNA. Thus, H/ACA RNPs are essential for three fundamental cellular processes: protein synthesis, mRNA splicing, and maintenance of genome integrity. Recently, great progress has been made toward understanding of the biogenesis, intracellular trafficking, structure, and function of H/ACA RNPs.