A plant snoRNP complex containing snoRNAs, fibrillarin, and nucleolin-like proteins is competent for both rRNA gene binding and pre-rRNA processing in vitro.
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.
Sáez-Vasquez
Julio
J
Laboratoire Génome et Développement des Plantes, UMR CNRS-IRD 5096, Université de Perpignan, France.
Caparros-Ruiz
David
D
Barneche
Fredy
F
Echeverría
Manuel
M
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001937:Brassica / Q000737:chemistry* / Q000378:metabolism; D002458:Cell Fractionation; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D009690:Nucleic Acid Conformation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012335:RNA, Ribosomal / Q000378:metabolism*; D020537:RNA, Small Nucleolar / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D031224:Raphanus / Q000737:chemistry / Q000378:metabolism; D020718:Ribonucleoproteins, Small Nucleolar / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D014158:Transcription, Genetic; D000097796:Nucleolin
D002868:Chromosomal Proteins, Non-Histone; D046911:Macromolecular Substances; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D010940:Plant Proteins; D012335:RNA, Ribosomal; D020537:RNA, Small Nucleolar; D016601:RNA-Binding Proteins; D020718:Ribonucleoproteins, Small Nucleolar; D000097796:Nucleolin; C047458:fibrillarin; C117377:ribonucleoprotein, U3 small nucleolar