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SYSTEMS BIOLOGY, MOLECULAR BIOLOGY, AND GENE REGULATION

Processing of a Dicistronic tRNA-snoRNA Precursor: Combined Analysis in Vitro and in Vivo Reveals Alternate Pathways and Coupling to Assembly of snoRNP¹

Laboratoire Génome et Développement des Plantes, Unité Mixte de Recherche 5096, Université de Perpignan Via Domitia-Centre National de la Recherche Scientifique-Institut de Recherche pour le Développement, 66860 Perpignan cedex, France (N.B., J.R., E.J., J.S.-V., M.E.); and Molekulare Botanik, Universität Ulm, 89069 Ulm, Germany (G.C., A.M.)

The C/D box small nucleolar RNAs (snoRNAs) represent an essential class of small nucleolar RNAs that guide 2'-O-Rib methylation of ribosomal RNAs and other RNAs in eukaryotes. In Arabidopsis (Arabidopsis thaliana), >100 C/D snoRNAs have been identified, most of them encoded by polycistronic gene clusters, but little is known on the factors controlling their biogenesis. Here, we focus on the identification of factors controlling the processing of tRNA-snoRNA dicistronic precursors (pre-tsnoRNA) synthesized by RNA polymerase III and producing tRNA^Gly and C/D snoR43. We produced radiolabeled RNA probes corresponding to different pre-tsnoRNA mutants to test their impact on processing in vitro by a recombinant tRNAse Z, the Arabidopsis endonuclease that processes the 3'end of tRNAs, and by nuclear extracts from cauliflower (Brassica oleracea) inflorescences that accurately process the pre-tsnoRNA. This was coupled to an in vivo analysis of the processing of tagged pre-tsnoRNA mutants expressed in Arabidopsis. Our results strongly implicate tRNase Z in endonucleolytic cleavage of the pre-tsnoRNA. In addition, they reveal an alternate pathway that could depend on a tRNA decay surveillance mechanism. Finally, we provide arguments showing that processing of pre-tsnoRNA, both in planta and by nuclear extracts, is coupled to the assembly of snoRNA with core proteins forming the functional snoRNP (for small nucleolar ribonucleoprotein complex).

² Present address: Institut de Génétique Humaine, Centre National de la Recherche Scientifique, Unité Propre de Recherche 1142, 141 rue de la cardonille, 34396 Montpellier cedex, France.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Manuel Echeverría (manuel.echeverria{at}univ-perp.fr).

www.plantphysiol.org/cgi/doi/10.1104/pp.109.137968

^* Corresponding author; e-mail manuel.echeverria{at}univ-perp.fr.

Received March 3, 2009; accepted April 29, 2009; published May 6, 2009.

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