RNomics in Archaea reveals a further link between splicing of archaeal introns and rRNA processing

The bulge–helix–bulge (BHB) motif recognised by the archaeal splicing endonuclease is also found in the long processing stems of archaeal rRNA precursors in which it is cleaved to generate pre-16S and pre-23S rRNAs. We show that in two species, Archaeoglobus fulgidus and Sulfolobus solfataricus, representatives from the two major archaeal kingdoms Euryarchaeota and Crenarchaeota, respectively, the pre-rRNA spacers cleaved at the BHB motifs surrounding pre-16S and pre-23S rRNAs subsequently become ligated. In addition, we present evidence that this is accompanied by circularisation of ribosomal pre-16S and pre-23S rRNAs in both species. These data reveal a further link between intron splicing and pre-rRNA processing in Archaea, which might reflect a common evolutionary origin of the two processes. One spliced RNA species designated 16S-D RNA, resulting from religation at the BHB motif of 16S pre-rRNA, is a highly abundant and stable RNA which folds into a three-stem structure interrupted by two single-stranded regions as assessed by chemical probing. It spans a region of the pre-rRNA 5′ external transcribed spacer exhibiting a highly conserved folding pattern in Archaea. Surprisingly, 16S-D RNA contains structural motifs found in archaeal C/D box small RNAs and binds to the L7Ae protein, a core component of archaeal C/D box RNPs. This supports the notion that it might have an important but still unknown role in pre-rRNA biogenesis or might even target RNA molecules other than rRNA.     

The evolutionarily conserved protein Las1 is required for pre-rRNA processing at both ends of ITS2

Ribosome synthesis entails the formation of mature rRNAs from long precursor molecules, following a complex pre-rRNA processing pathway. Why the generation of mature rRNA ends is so complicated is unclear. Nor is it understood how pre-rRNA processing is coordinated at distant sites on pre-rRNA molecules. Here we characterized, in budding yeast and human cells, the evolutionarily conserved protein Las1. We found that, in both species, Las1 is required to process ITS2, which separates the 5.8S and 25S/28S rRNAs. In yeast, Las1 is required for pre-rRNA processing at both ends of ITS2. It is required for Rrp6-dependent formation of the 5.8S rRNA 3' end and for Rat1-dependent formation of the 25S rRNA 5' end. We further show that the Rat1-Rai1 5'-3' exoribonuclease (exoRNase) complex functionally connects processing at both ends of the 5.8S rRNA. We suggest that pre-rRNA processing is coordinated at both ends of 5.8S rRNA and both ends of ITS2, which are brought together by pre-rRNA folding, by an RNA processing complex. Consistently, we note the conspicuous presence of ~7- or 8-nucleotide extensions on both ends of 5.8S rRNA precursors and at the 5' end of pre-25S RNAs suggestive of a protected spacer fragment of similar length.     

Intranuclear maturation pathways of rat liver ribosomal ribonucleic acids.

The maturation of pre-rRNA (precursor to rRNA)in liver nuclei is studied by agar/ureagel electrophoresis, kinetics of labelling in vivo with [14C] orotate and electron-microscopic observation of secondary structure of RNA molecules. (1) Processing starts from primary pre-rRNA molecules with average mol. wt. 4.6X10(6)(45S) containing the segments of both 28S and 18S rRNA. These molecules form a heterogeneous peak on electrophoresis. The 28S rRNA segment is homogeneous in its secondary structure. However, the large transcribed spacer segment (presumably at the 5'-end) is heterogeneous in size and secondary structure. A minor early labelled RNA component with mol.wt. about 5.8X10(6) is reproducibly found, but its role as a pre-rRNA species remains to be determined. (2) The following intermediate pre-rRNA species are identified: 3.25X10(6) mol.wt.(41S), a precursor common to both mature rRNA species ; 2.60X10(6)(36S) and 2.15X10(6)(32S) precursors to 28S rRNA; 1.05X10(6) (21S) precursor to 18S rRNA. The pre-rRNA molecules in rat liver are identical in size and secondary structure with those observed in other mammalian cells. These results suggest that the endonuclease-cleavage sites along the pre-rRNA chain are identical in all mammalian cells. (3) Labelling kinetics and the simultaneous existence of both 36S and 21S pre-rRNA reveal that processing of primary pre-rRNA in adult rat liver occurs simultaneously by at least two major pathways: (i) 45S leads to 41S leads to 32S+21S leads to 28S+18S rRNA and (ii) 45S leads to 41S leads to 36S+18S leads to 32S leads to 28S rRNA. The two pathways differ by the temporal sequence of endonuclease attack along the 41 S pre-rRNA chain. A minor fraction (mol.wt.2.9X10(6), 39S) is identified as most likely originating by a direct split of 28S rRNA from 45S pre-rRNA. These results show that in liver considerable flexibility exists in the order of cleavage of pre-rRNA molecules during processing.     

Nuclear export competence of pre-40S subunits in fission yeast requires the ribosomal protein Rps2

Ribosome biogenesis is an evolutionarily conserved pathway that requires ribosomal and nonribosomal proteins. Here, we investigated the role of the ribosomal protein S2 (Rps2) in fission yeast ribosome synthesis. As for many budding yeast ribosomal proteins, Rps2 was essential for cell viability in fission yeast and the genetic depletion of Rps2 caused a complete inhibition of 40S ribosomal subunit production. The pattern of pre-rRNA processing upon depletion of Rps2 revealed a reduction of 27SA₂ pre-rRNAs and the concomitant production of 21S rRNA precursors, consistent with a role for Rps2 in efficient cleavage at site A₂ within the 32S pre-rRNA. Importantly, kinetics of pre-rRNA accumulation as determined by rRNA pulse-chases assays indicated that a small fraction of 35S precursors matured into 20S-containing particles, suggesting that most 40S precursors were rapidly degraded in the absence of Rps2. Analysis of steady-state RNA levels revealed that some pre-40S particles were produced in Rps2-depleted cells, but that these precursors were retained in the nucleolus. Our findings suggest a role for Rps2 in a mechanism that monitors pre-40S export competence.     

Synthesis and maturation of ribosomal ribonucleic acids in isolated HeLa cell nuclei. A tracer study on the topology of the 45S precursor of ribosomal ribonucleic acids

The synthesis and processing of RNA by isolated HeLa cell nuclei was studied at low ionic strength in the presence of alpha-amanitin. The RNA polymerase reaction, with endogenous template and enzyme, rapidly reaches a plateau dependent on the amount of nuclei. Evidence is presented that incorporation of [(3)H]UMP proceeds only in growing RNA chains, whereas initiation of new RNA chains is arrested. The product formed contains all the main components of the 45S pre-rRNA (precursor of rRNA) maturation pathway (45S, 32S and 20S pre-rRNA; 28S and 18S rRNA). Most of the labelled material is in the mature rRNA components and their immediate precursors, even at very short times of incubation (2min). Small, but definite, 5S and 4S RNA peaks are also observed. At shorter incubation times a substantial amount of [(3)H]UMP is incorporated into RNA molecules in the 24S and 10-16S zones. This RNA material is considered to represent the non-conserved segments of 45S pre-rRNA in the process of nucleolytic degradation. A model for the tracer study of the topology of 45S pre-rRNA, on arrest of rRNA initiation, is discussed. The experimental evidence obtained supports the following structure of 45S pre-rRNA: 5'-end-28S rRNA unit-18S rRNA unit-nonconserved segment-3'-end.     

Imp3p and Imp4p, Two Specific Components of the U3 Small Nucleolar Ribonucleoprotein That Are Essential for Pre-18S rRNA Processing

The function of the U3 small nucleolar ribonucleoprotein (snoRNP) is central to the events surrounding pre-rRNA processing, as evidenced by the severe defects in cleavage of pre-18S rRNA precursors observed upon depletion of the U3 RNA and its unique protein components. Although the precise function of each component remains unclear, since U3 snoRNA levels remain unchanged upon genetic depletion of these proteins, it is likely that the proteins themselves have significant roles in the cleavage reactions. Here we report the identification of two previously undescribed protein components of the U3 snoRNP, representing the first snoRNP components identified by using the two-hybrid methodology. By screening for proteins that physically associate with the U3 snoRNP-specific protein, Mpp10p, we have identified Imp3p (22 kDa) and Imp4p (34 kDa) (named for interacting with Mpp10p). The genes encoding both proteins are essential in yeast. Genetic depletion reveals that both proteins are critical for U3 snoRNP function in pre-18S rRNA processing at the A0, A1, and A2 sites in the pre-rRNA. Both Imp proteins associate with Mpp10p in vivo, and both are complexed only with the U3 snoRNA. Conservation of RNA binding domains between Imp3p and the S4 family of ribosomal proteins suggests that it might associate with RNA directly. However, as with other U3 snoRNP-specific proteins, neither Imp3p nor Imp4p is required for maintenance of U3 snoRNA integrity. Imp3p and Imp4p are therefore novel protein components specific to the U3 snoRNP with critical roles in pre-rRNA cleavage events.