Functional contacts with a range of splicing proteins suggest a central role for Brr2p in the dynamic control of the order of events in spliceosomes of Saccharomyces cerevisiae.

Mapping of functional protein interactions will help in understanding conformational rearrangements that occur within large complexes like spliceosomes. Because the U5 snRNP plays a central role in pre-mRNA splicing, we undertook exhaustive two-hybrid screening with Brr2p, Prp8p, and other U5 snRNP-associated proteins. DExH-box protein Brr2p interacted specifically with five splicing factors: Prp8p, DEAH-box protein Prp16p, U1 snRNP protein Snp1p, second-step factor Slu7p, and U4/U6.U5 tri-snRNP protein Snu66p, which is required for splicing at low temperatures. Co-immunoprecipitation experiments confirmed direct or indirect interactions of Prp16p, Prp8p, Snu66p, and Snp1p with Brr2p and led us to propose that Brr2p mediates the recruitment of Prp16p to the spliceosome. We provide evidence that the prp8-1 allele disrupts an interaction with Brr2p, and we propose that Prp8p modulates U4/U6 snRNA duplex unwinding through another interaction with Brr2p. The interactions of Brr2p with a wide range of proteins suggest a particular function for the C-terminal half, bringing forward the hypothesis that, apart from U4/U6 duplex unwinding, Brr2p promotes other RNA rearrangements, acting synergistically with other spliceosomal proteins, including the structurally related Prp2p and Prp16p. Overall, these protein interaction studies shed light on how splicing factors regulate the order of events in the large spliceosome complex.

Variable region V1 of Saccharomyces cerevisiae 18S rRNA participates in biogenesis and function of the small ribosomal subunit.

The role of helix 6, which forms the major portion of the most 5'-located expansion segment of Saccharomyces cerevisiae 18S rRNA, was studied by in vivo mutational analysis. Mutations that increased the size of the helical part and/or the loop, even to a relatively small extent, abolished 18S rRNA formation almost completely. Concomitantly, 35S pre-rRNA and an abnormal 23S precursor species accumulated. rDNA units containing these mutations did not support cell growth. A deletion removing helix 6 almost completely, on the other hand, had a much less severe effect on the formation of 18S rRNA, and cells expressing only the mutant rRNA remained able to grow, albeit at a much reduced rate. Disruption of the apical A.U base pair by a single point mutation did not cause a noticeable reduction in the level of 18S rRNA but did result in a twofold lower growth rate of the cells. This effect could not be reversed by introduction of a second point mutation that restores base pairing. We conclude that both the primary and the secondary structure of helix 6 play an important role in the formation and the biological function of the 40S subunit.

Processing of eukaryotic pre-rRNA: the role of the transcribed spacers.

The 17-18S, 5.8S, and 25-28S rRNA species of eukaryotic cells are produced by a series of nucleolytic reactions that liberate the mature rRNAs from the large primary precursor transcript synthesized by RNA polymerase 1. Whereas the order of the cleavage reactions has long been established, until recently little information was available on their molecular details, such as the nature of the proteins, including the nucleolytic enzymes, involved and the signals directing the processing machinery to the correct sites. This situation is now rapidly changing, in particular where yeast is concerned. The use of recently developed systems for in vivo mutational analysis of yeast rDNA has considerably enhanced our knowledge of cis-acting structural features within the pre-rRNA, in particular the transcribed spacer sequences, that are critical for correct and efficient removal of these spacers. The same systems also allow a link to be forged between trans-acting processing factors and these cis-acting elements. In this paper, we will focus predominantly on the nature and role of the cis-acting processing elements as identified in the transcribed spacer regions of Saccharomyces cerevisiae pre-rRNA.

Evolutionarily conserved structural elements are critical for processing of Internal Transcribed Spacer 2 from Saccharomyces cerevisiae precursor ribosomal RNA.

Structural features of Internal Transcribed Spacer 2 (ITS2) important for the correct and efficient removal of this spacer from Saccharomyces cerevisiae pre-rRNA were identified by in vivo mutational analysis based upon phylogenetic comparison with its counterparts from four different yeast species. Compatibility between ITS2 structure and the S. cerevisiae processing machinery was found to have been maintained over only a short evolutionary distance, in contrast to the situation for ITS1. Nevertheless, cis-acting elements required for correct and efficient processing are confined predominantly to those regions of the spacer that show the highest degree of evolutionary conservation. Mutation or deletion of each of these regions severely reduced production of mature 26 S, but not 17 S rRNA, mainly by impeding processing of the 29 SB precursor. In some cases, however, conversion of 29SA into 29 SB pre-rRNA also appeared to be affected. Deletion of non-conserved segments, on the other hand, caused little or no disturbance in processing. Surprisingly, some combinations of such individually neutral deletions had a severe negative effect on the removal of ITS2, suggesting a requirement for a higher-order structure of ITS2. Finally, even structural alterations of ITS2 that did not noticeably affect processing, significantly reduced the growth rate of cells that exclusively express the mutant rDNA units. We take this as further evidence for a direct role of ITS2 in the formation of fully functional 60 S ribosomal subunits.

Separate structural elements within internal transcribed spacer 1 of Saccharomyces cerevisiae precursor ribosomal RNA direct the formation of 17S and 26S rRNA.

Structural features of Internal Transcribed Spacer 1 (ITS1) that direct its removal from Saccharomyces cerevisiae pre-rRNA during processing were identified by an initial phylogenetic approach followed by in vivo mutational analysis of specific structural elements. We found that S. cerevisiae ITS1 can functionally be replaced by the corresponding regions from the yeasts Torulaspora delbrueckii, Kluyveromyces lactis and Hansenula wingei, indicating that structural elements required in cis for processing are evolutionarily conserved. Despite large differences in size, all ITS1 regions conform to the secondary structure proposed by Yeh et al. [Biochemistry 29 (1990) 5911-5918], showing five domains (I-V; 5'-->3') of which three harbour an evolutionarily highly conserved element. Removal of most of domain II, including its highly conserved element, did not affect processing. In contrast, highly conserved nucleotides directly downstream of processing site A2 in domain III play a major role in production of 17S, but not 26S rRNA. Domain IV and V are dispensable for 17S rRNA formation although an alternative, albeit inefficient, processing route to mature 17S rRNA may be mediated by a conserved region in domain IV. Each of these two domains is individually sufficient for efficient production of 26S rRNA, suggesting two independent processing pathways. We conclude that ITS1 is organized into two functionally and structurally distinct halves.

Isolation and sequencing of Escherichia coli gene proP reveals unusual structural features of the osmoregulatory proline/betaine transporter, ProP.

Transporters encoded in genetic loci putP, proP and proU mediate proline and/or betaine accumulation by Escherichia coli K-12. The ProP and ProU systems are osmoregulatory. Activation of ProP in response to hyperosmotic stress has been demonstrated both in vivo and in vitro. It therefore serves as a model experimental system for the analysis of osmosensory and osmoregulatory mechanisms. We developed methodologies which will facilitate the identification of proline transporter genes by functional complementation of putP proP proU bacteria. E. coli gene proP was isolated and located within a chromosomal DNA fragment. Deletion, complementation and sequence analysis revealed putative promoter and transcription termination signals flanking a 1500 base-pair open reading frame. The predicted 55 kDa ProP protein was hydrophobic. In vitro expression of proP yielded a protein whose apparent molecular mass was determined to be 42 kDa by polyacrylamide gel electrophoresis under denaturing conditions. Database searches and cluster analysis defined relationships among the ProP sequence and those of integral membrane proteins that comprise a transporter superfamily. Members of the superfamily catalyze facilitated diffusion or ion linked transport of organic solutes in prokaryotes and eukaryotes. Multiple alignment revealed particularly close correspondence among the ProP protein, citrate transporters from E. coli and Klebsiella pneumoniae and an alpha-ketoglutarate transporter from E. coli. The predicted ProP sequence differed from those closely similar sequences in possessing an extended central hydrophilic loop and a carboxyl terminal extension. Unlike other protein sequences within the transporter superfamily, the carboxyl terminal extension of ProP was strongly predicted to participate in formation of an alpha-helical coiled coil. These data suggest that the ProP protein catalyzes solute-ion cotransport. Its unusual structural features may be related to osmoregulation of its activity.

Functional analysis of internal transcribed spacer 2 of Saccharomyces cerevisiae ribosomal DNA.

Using the previously described "tagged ribosome" (pORCS) system for in vivo mutational analysis of yeast rDNA, we show that small deletions in the 5'-terminal portion of ITS2 completely block maturation of 26 S rRNA at the level of the 29 SB precursor (5.8 S rRNA-ITS2-26 S rRNA). Various deletions in the 3'-terminal part, although severely reducing the efficiency of processing, still allow some mature 26 S rRNA to be formed. On the other hand, none of the ITS2 deletions affect the production of mature 17 S rRNA. Since all of the deletions severely disturb the recently proposed secondary structure of ITS2, these findings suggest an important role for higher order structure of ITS2 in processing. Analysis of the effect of complete or partial replacement of S. cerevisiae ITS2 with its counterpart sequences from Saccharomyces rosei or Hansenula wingei, points to helix V of the secondary structure model as an important element for correct and efficient processing. Direct mutational analysis shows that disruption of base-pairing in the middle of helix V does not detectably affect 26 S rRNA formation. In contrast, introduction of clustered point mutations at the apical end of helix V that both disrupt base-pairing and change the sequence of the loop, severely reduces processing. Since a mutant containing only point mutations in the sequence of the loop produces normal amounts of mature 26 S rRNA, we conclude that the precise (secondary and/or primary) structure at the lower end of helix V, but excluding the loop, is of crucial importance for efficient removal of ITS2.