A Plant snoRNP Complex Containing snoRNAs, Fibrillarin, and Nucleolin-Like Proteins Is Competent for both rRNA Gene Binding and Pre-rRNA Processing In Vitro

Abstract

In eukaryotes the primary cleavage of the precursor rRNA (pre-rRNA) occurs in the 5 external transcribed spacer (5ETS). In Saccharomyces cerevisiae and animals this cleavage depends on a conserved U3 small nucleolar ribonucleoprotein particle (snoRNP), including fibrillarin, and on other transiently associated proteins such as nucleolin. This large complex can be visualized by electron microscopy bound to the nascent pre-rRNA soon after initiation of transcription. Our group previously described a radish rRNA gene binding activity, NF D, that specifically binds to a cluster of conserved motifs preceding the primary cleavage site in the 5ETS of crucifer plants including radish, cauliflower, and Arabidopsis thaliana (D. Caparros-Ruiz, S. Lahmy, S. Piersanti, and M. Echeverria, Eur. J. Biochem. 247:981-989, 1997). Here we report the purification and functional characterization of NF D from cauliflower inflorescences. Remarkably NF D also binds to 5ETS RNA and accurately cleaves it at the primary cleavage site mapped in vivo. NF D is a multiprotein factor of 600 kDa that dissociates into smaller complexes. Two polypeptides of NF D identified by microsequencing are homologues of nucleolin and fibrillarin. The conserved U3 and U14 snoRNAs associated with fibrillarin and required for early pre-rRNA cleavages are also found in NF D. Based on this it is proposed that NF D is a processing complex that assembles on the rDNA prior to its interaction with the nascent pre-rRNA.The rRNAs 18S, 5.8S, and 25S are encoded by tandemly repeated single transcriptional units. Transcription of these units by RNA polymerase I (RNA Pol I) produces a primary transcript (pre-rRNA) containing the rRNA flanked by external spacer sequences (ETS) and internal spacer sequences. The pre-rRNA is then subjected to a complex maturation process that involves the accurate removal of the spacers and the modification of numerous rRNA residues (49). In vivo, all these events are accomplished by large small nucleolar ribonucleoprotein particle (snoRNP) complexes that transiently interact with the pre-rRNA in the nucleolus (47).One of the earliest processing events on the pre-rRNA is an endonucleolytic cut in the 5ЈETS upstream from the 18S rRNA. This primary pre-RNA cleavage is conserved in all eukaryotes, but its position within the 5ЈETS is distinct in each species. In Saccharomyces cerevisiae it occurs at site A0, located 90 nucleotides upstream from the 5Ј end of the 18S rRNA (49). Genetic studies have shown that A0 cleavage depends on the U3 snoRNP, a large complex containing the C/D snoRNA U3 and associated nucleolar proteins (16). This implicates a basepair interaction of U3 with the 5ЈETS that may stabilize or promote a pre-RNA structure required for processing and subsequent production of 18S rRNA (5, 6). The U3 snoRNP proteins, including Nop1p (fibrillarin in vertebrates), and three other core proteins associated with all C/D snoRNAs are essential for this cleavage. In addition, there are proteins which are specific to U3 snoRNP and are not fou...

Pol I Transcription and Pre-rRNA Processing Are Coordinated in a Transcription-dependent Manner in Mammalian Cells

“…These observations indicate that transcription factors can be mobilized to the site of transcription separately from pre-rRNA processing factors, which are recruited only in the presence of the specific substrate. This suggests that the association of pre-rRNA processing factors to the newly transcribed rRNA is sequential and is on anneeded basis.Our findings that pol I transcription factors interact with pre-rRNA processing factors are consistent with recent studies in nonmammalian systems that found transcription factors in the same complex with pre-rRNA processing factors (Fath et al, 2000;Gallagher et al, 2004) and coordination between the two processes (Veinot-Drebot et al, 1988;Caparros-Ruiz et al, 1997;Dragon et al, 2002;Gallagher et al, 2004;Osheim et al, 2004;Saez-Vasquez et al, 2004). Studies in yeast showed that most nascent pre-rRNA transcripts no longer contain 5Ј external transcribed spacer sequences (Veinot-Drebot et al, 1988) and that the initial steps of pre-rRNA processing take place before the completion of pre-rRNA transcription (Dragon et al, 2002;Osheim et al, 2004).…”

“…The coordination between transcription and pre-rRNA processing could be facilitated through at least two different mechanisms. The transcription and processing factors could be corecruited to the site of transcription in a large complex, as the pol II system is coordinated, which is an idea that is supported by a number of published studies (Fath et al, 2000;Saez-Vasquez et al, 2004). Alternatively, processing factors could be recruited to the sites of transcription only when the nascent pre-rRNA substrate becomes available.…”

“…Specifically, it is not clear whether the pol I transcription and pre-rRNA processing factors are corecruited in the same complex to the promoter or if the processing factors are only recruited in the presence of nascent pre-rRNA. Studies in plants show that a complex containing a snoRNP that includes fibrillarin and nucleolin-like proteins is competent for both rDNA binding (in the absence of transcription) and pre-rRNA processing in vitro, suggesting that they are in the same complex before rDNA binding (Caparros-Ruiz et al, 1997;Saez-Vasquez et al, 2004). Additionally, a nucleolar RNP complex containing pol I transcription and pre-rRNA processing factors has been isolated in yeast cells that were pol I deficient or transcriptionally incompetent (Fath et al, 2000).…”

Characterization of AtNUC-L1 Reveals a Central Role of Nucleolin in Nucleolus Organization and Silencing of AtNUC-L2 Gene in Arabidopsis

“…Primer extension analysis was performed using total RNA and specific 5Ј-end labeled primers as previously described (Saez-Vasquez et al, 2004). Primers used were tis for detection of primary pre-rRNA precursor (ϩ104 nucleotides from transcription initiation site [TIS]) and p (ϩ1362 nucleotides from TIS) for detection of pre-rRNA cleaved at the P site ( Figure 9A).…”

“…The extracts were cleared by centrifugation at 13,000 ϫ g for 15 min and conserved at Ϫ80°C. SDS-PAGE, and Western blot was performed as described previously (Saez-Vasquez et al, 2004). The membranes were hybridized with a 1:10,000 dilution of ␣-NUC1 or with a 1:5000 dilution of ␣-NUC2.…”