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 (5'ETS). 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 5'ETS 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 5'ETS 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.

FIG. 1.

Primary pre-rRNA cleavage site in the 5′ETS of crucifer plants. (A) Mapping by primer extension of pre-rRNA cleavages in the 5′ETSs of radish (Raphanus sativus), cauliflower (Brassica oleracea), and Arabidopsis (A. thaliana). Reverse transcriptions were performed on total seedling RNAs from primers p1 to p4 as indicated. P3 is complementary to both the radish and the cauliflower 5′ETS sequences. Open and closed arrows indicate the TIS and the primary processing site (P), respectively. CTGA are 5′ETS DNA sequences made from corresponding primers (p2 for radish). The scheme of the 5′ETS with the position of TIS, the cleavage signal P, and the primers used for mapping is shown to scale. Nucleotides are numbered from the TISs that were previously mapped (7, 13, 25). (B) Mapping of the primary processing site in the radish 5′ETS by RNase protection analysis. RNaseA/T1 protection was done with a radiolabeled RNA probe complementary to the 5′ETS radish sequence, including the P site detected by primer extension. The riboprobe sequence complementary to the 5′ETS is indicated. Eight additional nucleotides from vector sequences are indicated by thin lines at the ends of the riboprobe. The expected sizes of protected fragments are shown. The assay was carried out with the indicated amount of total RNA from radish seedlings or yeast tRNA as indicated. A control lane loaded with the untreated riboprobe is shown (lane 0). Following the RNase reaction the protected fragments were analyzed in a 6% sequencing gel. The sizes of protected bands evaluated from a marker ladder run in parallel (not shown) are indicated on the right of the gel.

FIG. 2.

The A¹²³B cluster and predicted structures flanking the primary processing site in the 5′ETSs of crucifers. (A) Alignment of the sequence encompassing the primary pre-rRNA cleavage site and the A¹²³B cluster in crucifers. The A¹, A², A³, B, and P motifs are overlined. Rs, R. sativus (13); Bo, B. oleracea (7); Br, Brassica rapa (12); Bj, Brassica juncea (26); At, A. thaliana (25). EMBL accession numbers: Rs, Z11677; Bo, X60324; Br, S78172; Bj, X73032; At, X52631. (B) The hairpin structures predicted downstream from the P site are shown for radish and Arabidopsis. Secondary structures were predicted by using Mfold (50).

FIG. 3.

NF D and NF B activities in crucifer extracts. (A) Diagram of the rDNA probe A¹²³BP used in the EMSA to detect NF D and NF B. The probe encompasses genomic 5′ETS rDNA nucleotides from +103 to +205. It has no additional sequences except for flanking restriction sites (11). The nucleotide sequence shown is the rDNA binding site of NF D mapped by DNase I footprinting (data from reference 11). (B) EMSA with the A¹²³BP probe and the indicated amounts of whole-cell extracts (WCE) from radish seedlings or cauliflower inflorescences. Arrows on the right show NF D and NF B_1-2 complexes.

FIG. 4.

Purification of NF D. (A) Scheme of purification of NF D. Details are given in Materials and Methods. (B) Sephacryl S300 elution profile of NF D activity detected by EMSA with the rDNA A¹²³BP probe. Pool indicates fractions loaded on the oliA DNA-Sepharose column. (C) oliA DNA-Sepharose elution profile of NF D activity detected with the rDNA A¹²³BP probe. (D) Protein profile of pure NF D separated by SDS-PAGE and revealed by silver staining. The asterisks indicate bands identified by Western blotting as AtNuc-L1 and AtFib.

FIG. 5.

NF D binds to the 5′ETS RNA. (A) The RNA probe rA¹²³BPH, encompassing nucleotides +103 to +317 from the radish 5′ETS, is shown by the continuous line. The A¹²³B motifs, the P site, and the predicted hairpin are indicated. (B) The rRNA binding activity in the oliA DNA-Sepharose-eluting fractions detected by EMSA with the rRNA A¹²³BPH probe. Fractions are from the same column analyzed for Fig. 4C. (C) Competition for binding of NF D to rDNA probe A¹²³BP with increasing amounts of unlabeled rDNA A¹²³BP or rA¹²³BPH as indicated.

FIG. 6.

AtNuc-L1 and AtFib are components of NF D. (A) Alignment of sequences from peptides 1 to 3 derived from the 67- and 31-kDa polypeptides to predicted AtNuc-L1 (GenBank accession no. AC084414), AtNuc-L2 (GenBank accession no. AP001303), and AtFib1 and AtFib2 (4). Protein structures and domains are schematized to scale. The positions of similar peptide sequences of Arabidopsis proteins in predicted AtNuc-L1, AtNuc-L2, and AtFib1/AtFib 2 are shown. Asterisks indicate changes between the cauliflower and Arabidopsis peptide sequences. RRM, RNA recognition motif (1); RGG, arginine-glycine-rich domain. The horizontal lines indicated by α-AtNuc-L1 and α-AtFib show the protein regions that were overexpressed and used as antigens to raise specific antibodies. (B) Western blot of NF D and NF B fractions with antibodies against AtNuc-L1 or AtFib. Total proteins from purified NF D or NF B separated fractions as indicated were used for Western blotting (see Materials and Methods) with antibodies directed against AtNuc-L1 or AtFib. The purified NF B fraction corresponds to a pool of fractions eluting below 250 kDa in a Sephacryl S300 column and subsequently purified by oliB DNA chromatography as previously reported (11). (C) Effects of anti-AtNuc-L1 (α-AtNuc-L1) and α-AtFib antibodies in DNA binding activity of NF D. The purified NF D was subjected to immunodepletion (see Materials and Methods) with antibody α-AtNuc-L1, α -AtFib, or α -RPL13 or PI (preimmune serum). After treatment, immunodepleted extracts were assayed by EMSA with the rDNA A¹²³BP probe.

FIG. 7.

Identification of C/D snoRNAs in NF D by RT-PCR. Specific primers were designed to detect U3, U14, R82, and U6 based on Arabidopsis gene sequences (see Materials and Methods). RT-PCR was carried out with the corresponding primer pairs and either total RNA from cauliflower inflorescence or RNA extracted from purified NF D fraction as indicated. In control reaction mixtures reverse transcriptase was omitted from the RT-PCR assay, as indicated.

FIG. 8.

Specific cleavage of rA¹²³BPH RNA by NF D fraction. (A) Fractions of the peak of NF D activity eluting from oliA DNA chromatography (shown in Fig. 4C) were incubated in a cleavage reaction buffer with RNA probe rA¹²³BPH (see Materials and Methods). The open arrowhead indicates full-length RNA probe. Closed arrows indicate cleavage products. Size markers are indicated on the left. (B) Cleavage reaction was carried out with unlabeled rA¹²³BPH RNA substrate in the presence (lane 8) or absence (lane 7) of purified NF D (see Materials and Methods). After incubation, products of the reaction were analyzed by primer extension using primer p3 (shown in Fig. 1). The cleavage site on the pre-rRNA produced in vivo was mapped with the same primer on total RNA extracted from radish seedlings (lane 6). A control was done without RNA added to the reaction mixture (lane 5). CTAG indicates the DNA sequence of the radish 5′ETS made with the same primer. Closed arrows indicate cleavage signals according to their intensities; closed arrowheads indicate the positions of the cleavage signals on the sequence.