Functional interaction of a novel 15.5kD [U4/U6middle dotU5] tri-snRNP protein with the 5' stem-loop of U4 snRNA

Nottrott, Stephanie

doi:10.1093/emboj/18.21.6119

Cited by 183 publications

(262 citation statements)

References 63 publications

(96 reference statements)

Supporting

Mentioning

250

Contrasting

Unclassified

Order By: Relevance

“…The human 15.5-kDa protein has been shown to interact directly with the 5Ј stem loop of U4 snRNA and a similar structural motif generated by the interaction between the C and D box sequences in U3, U8, and U14 snoRNAs (18). Archaeal sRNAs exhibit a bipartite structure with well-defined box C and D sequences near their respective 5Ј and 3Ј ends and related CЈ and DЈ sequences near the center of the molecules.…”

Section: Resultsmentioning

confidence: 99%

“…Replacement of two amino acids in this motif (A157V and P219S) in the S. cerevisiae Nop1p protein resulted in temperature sensitivity and a dramatic reduction in methylation of nascent rRNA transcripts under restrictive conditions (17). The 15.5-kDa protein is a component of both the C͞D box snoRNPs and the U4͞U6⅐U5 tri small nuclear RNP (snRNP), and belongs to a larger family of related RNA-binding proteins that include human L7a and S12 and yeast L30 ribosomal proteins (14,18,19). These proteins bind to a common RNA structural motif consisting of an internal purine-rich asymmetric loop with an unusual fold.…”

mentioning

confidence: 99%

See 1 more Smart Citation

In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex

Omer

Ziesche²,

Ebhardt³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

176

223

View full text Add to dashboard Cite

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)...

show abstract

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex

Omer

Ziesche²,

Ebhardt³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

176

223

View full text Add to dashboard Cite

show abstract

“…Hydroxyl Radical Footprinting-U4 snRNA (0.6 pmol; prepared by transcription in vitro (22)) was incubated for 1 h at 4°C with 25 pmol of recombinant 15.5K and/or 50 pmol of hPrp31 proteins in a final volume of 40 l buffer A (20 mM HEPES-KOH (pH 7.9), 150 mM KCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.1% Triton X-100). Ribose cleavage of the RNA backbone was initiated by hydroxyl radicals generated with H 2 O 2 and Fe(II)-EDTA as described (22), and cleavage products were analyzed by primer extension using a 32 P-labeled oligonucleotide complementary to nucleotides 65-82 of human U4 snRNA as before (22).…”

Section: Methodsmentioning

confidence: 99%

RNA Structural Requirements for the Association of the Spliceosomal hPrp31 Protein with the U4 and U4atac Small Nuclear Ribonucleoproteins

Schultz¹,

Nottrott²,

Hartmuth

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

The kink-turn, a stem I-internal loop-stem II structure of the 5 stem-loop of U4 and U4atac small nuclear (sn) RNAs bound by 15.5K protein is required for binding of human Prp31 protein (hPrp31) during U4 and U4atac snRNP assembly. In box C/D snoRNPs a similar kink-turn with bound 15.5K protein is required for selective binding of proteins NOP56 and NOP58. Here we analyzed RNA structural requirements for association of hPrp31 with U4 snRNP in vitro by hydroxyl radical footprinting. hPrp31 induced protection of the terminal penta-loop, as well as of stems I and II flanking the kink-turn. Similar protection was found with U4/U6 snRNA duplex prebound with 15.5K protein. A detailed mutational analysis of the U4 snRNA elements by electrophoretic mobility shift analysis revealed that stem I could not be shortened, although it tolerated sequence alterations. However, introduction of a third Watson-Crick base pair into stem II significantly reduced hPrp31 binding. While stem I of U4atac snRNA showed relaxed binding requirements, its stem II requirements were likewise restricted to two base pairs. In contrast, as shown previously, stem II of the kink-turn motif in box C/D snoRNAs is comprised of three base pairs, and NOP56 and NOP58 require a G-C pair at the central position. This indicates that hPrp31 binding specificity is achieved by the recognition of the two base pair long stem II of the U4 and U4atac snRNAs and suggests how discrimination is achieved by RNA structural elements during assembly of U4/U6 and U4atac/ U6atac snRNPs and box C/D snoRNPs.The processing of pre-mRNA in the nucleus is catalyzed by a large RNA-protein complex, the spliceosome (1-3). The spliceosome comprises, apart from the pre-mRNA substrate, the U1, U2, U4, U5, and U6 snRNPs, 4 as well as numerous splicing factors, that is, proteins that do not make up an integral part of the snRNPs. Each snRNP is a stable complex, consisting of a single RNA molecule and various proteins, of which some (the so-called Sm proteins) are common to all snRNPs, while others are specific to a particular snRNP (2). The individual snRNPs interact with one another in a dynamic manner during the splicing cycle. For example, at the beginning of this cycle the U4 snRNP and the U6 snRNP are associated firmly with one another by two extended stretches of RNA-RNA base pairs. Two intermolecular helices of the U4/U6 snRNA are separated by an intramolecular 5Ј-terminal hairpin loop (5Ј stem-loop) of the U4 snRNA (4 -7). The 5Ј-terminal stem-loop of the U4 snRNA (Fig. 1B) consists of a stem I, an intramolecular loop and then a stem II. The latter ends in a pentanucleotide loop ("penta-loop"). Thus at this stage the U4 and U6 snRNP form a single particle, the U4/U6 di-snRNP. This complex, in turn, associates with the U5 snRNP and thus the U4/U6⅐U5 tri-snRNP is formed, which is integrated as such into the precatalytic spliceosome. During the rearrangement of the fully assembled spliceosome to its catalytically active state, the intermolecular base pairing between the U4 and U6 sn...

show abstract

“…The most common K-turn binding protein is the ribosomal L7Ae and related proteins. These form a family of RNA-binding proteins including the eukaryotic and archaeal proteins L7Ae, L30e, and S12e (Koonin et al 1994), the yeast Nhp2 and Snu13p proteins, and the human 15.5-kDa protein (Nottrott et al 1999). K-turns are found in the box C/D and H/ACA guide snoRNA species that direct site-specific methylation and pseudouridylation, respectively, of RNA in archaea and eukaryotes (Kiss-Laszlo et al 1996;Ganot et al 1997).…”

Section: Introductionmentioning

confidence: 99%

“…These modifications require the formation of a nucleoprotein complex, the assembly of which requires initial binding of an L7Ae-related protein to the K-turn motif. The 15.5-kDa protein also binds a K-turn within the U4 stemloop within the U4-U6.U5 tri-snRNP (Nottrott et al 1999).…”

Section: Introductionmentioning

confidence: 99%

The role of specific 2′-hydroxyl groups in the stabilization of the folded conformation of kink-turn RNA

Liu

Lilley²

2006

RNA

137

View full text Add to dashboard Cite

The role of 29-hydroxyl groups in stabilizing the tightly kinked geometry of the kink-turn (K-turn) has been investigated. Individual 29-OH groups have been removed by chemical synthesis, and the kinking of the RNA has been studied by gel electrophoresis and fluorescence resonance energy transfer. The results have been analyzed by reference to a database of 11 different crystallographic structures of K-turns. The potential hydrogen bonds fall into several classes. The most important are those in the core of the turn and ribose-phosphate interactions around the bulge. Of these the single most important hydrogen bond is one donated from the 29-OH of the 59 nucleotide of the bulge to the N1 of the adenine of the kink-proximal A d G pair. This is present in all known K-turn structures, and removal of the 29-OH completely prevents metal ion-induced folding. Hydrogen bonds formed in the minor grooves of the helical stems are less important, and removal of the participating 29-OH groups leads to reduced impairment of folding. These interactions are generally more polymorphic, and hydrogen bonds probably form where possible, as permitted by the global structure.

show abstract

Functional interaction of a novel 15.5kD [U4/U6middle dotU5] tri-snRNP protein with the 5' stem-loop of U4 snRNA

Cited by 183 publications

References 63 publications

In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex

In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex

RNA Structural Requirements for the Association of the Spliceosomal hPrp31 Protein with the U4 and U4atac Small Nuclear Ribonucleoproteins

The role of specific 2′-hydroxyl groups in the stabilization of the folded conformation of kink-turn RNA

Contact Info

Product

Resources

About