Processing of a dicistronic tRNA-snoRNA precursor: combined analysis in vitro and in vivo reveals alternate pathways and coupling to assembly of snoRNP.

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).