Trypanosoma brucei 5'ETS A'-cleavage is directed by 3'-adjacent sequences, but not two U3 snoRNA-binding elements, which are all required for subsequent pre-small subunit rRNA processing events.

Trypanosoma brucei pre-rRNA processing commences by cleavage near the 5' end of 5.8 S sequences. The 5' external transcribed spacer (5'ETS) is removed from pre-small subunit (SSU) rRNAs by sequential cleavages at internal A' and A0 sites, and A1 at the 5' end of SSU rRNA. The A' and A0 sites positionally resemble the U3 small nucleolar RNA-dependent, primary pre-rRNA cleavages of vertebrates and yeast, respectively. Uniquely in T. brucei, two U3-crosslinkable 5'ETS sites are essential for SSU rRNA production: site1b is novel in its 3' location to the A' site, and site3 lies upstream of A0 in a position analogous to the yeast U3-binding site. Here, in vivo analysis of mutated 5'ETS sequences shows that sequences 5' to the A' site are not needed for A' cleavage or SSU rRNA production. A' cleavage is linked to, but is not sufficient to trigger, downstream pre-SSU rRNA processing events. These events require an intact 11 nt sequence, 3'-adjacent to A', which directs efficient and accurate A' cleavage. Neither the A' nearby site1b nor the site3 U3-binding elements affect A' processing, yet each is required for A0 and A1 cleavage, and SSU rRNA production. The same U3 3' hinge bases evidently bind a core element, UGUu/gGGU, within site1a and site3; the U3-site1b interaction is less reliant on base-pairing than the U3-site3 interaction. As yeast U3 5' hinge bases pair to 5'ETS sequences, it is clear that distinct U3 hinge regions can interact at both novel and related 5'ETS sites to promote 3'-proximal 5'ETS processing events in diverse organisms. The T. brucei data fit a model wherein processing factors assemble at the 5'ETS site1a to affect A' cleavage and stabilize a U3-site1b complex, which may work in concert with the downstream U3-site3 complex to assist processing events leading to ribosomal SSU production.     

Phylogenetic analysis of ribosomal RNA operons from uncultivated coastal marine bacterioplankton

Analyses of small subunit ribosomal RNA genes (SSU rDNAs) have significantly influenced our understanding of the composition of aquatic microbial assemblages. Unfortunately, SSU rDNA sequences often do not have sufficient resolving power to differentiate closely related species. To address this general problem for uncultivated bacterioplankton taxa, we analysed and compared sequences of polymerase chain reaction (PCR)-generated and bacterial artificial chromosome (BAC)-derived clones that contained most of the SSU rDNAs, the internal transcribed spacer (ITS) and the large subunit ribosomal RNA gene (LSU rDNA). The phylogenetic representation in the rRNA operon PCR library was similar to that reported previously in coastal bacterioplankton SSU rDNA libraries. We observed good concordance between the phylogenetic relationships among coastal bacterioplankton inferred from SSU or LSU rDNA sequences. ITS sequences confirmed the close intragroup relationships among members of the SAR11, SAR116 and SAR86 clades that were predicted by SSU and LSU rDNA sequence analyses. We also found strong support for homologous recombination between the ITS regions of operons from the SAR11 clade.     

The novel organization and complete sequence of the ribosomal RNA gene of Nosema bombycis

We present here for the first time the complete DNA sequence data (4301bp) of the ribosomal RNA (rRNA) gene of the microsporidian type species, Nosema bombycis. Sequences for the large subunit gene (LSUrRNA: 2497bp, GenBank Accession No. ), the internal transcribed spacer (ITS: 179bp, GenBank Accession No. ), the small subunit gene (SSUrRNA: 1232bp), intergenic spacer (IGS: 279bp), and 5S region (114bp) are also given, and the secondary structure of the large subunit is discussed. The organization of the N. bombycis rRNA gene is LSUrRNA-ITS-SSUrRNA-IGS-5S. This novel arrangement, in which the LSU is 5' of the SSU, is the reverse of the organizational sequence (i.e., SSU-ITS-LSU) found in all previously reported microsporidian rRNAs, including Nosema apis. This unique character in the type species may have taxonomic implications for the members of the genus Nosema.     

Organization of ribosomal RNA gene repeats of the mouse.

The organization of the ribosomal RNA (rRNA) genes of the mouse was determined by Southern blot hybridization using cloned rDNA fragments as probes, which could encompass the entire spacer region between two rRNA gene regions. The rRNA genes are organized into tandem repeats of nearly uniform length of about 44 kb. The heterogeneity detected in the nontranscribed spacer appears to be caused by its sequence rather than its length difference. At least three kinds of repetitive sequences are present in the non-transcribed spacer region; two of them are located 13 kb upstream from the 5'-end of 18S RNA gene and the other located 1 to 4 kb downstream from the 3'-end of 28S RNA gene.