Structure-function analysis of yeast RNA debranching enzyme (Dbr1), a manganese-dependent phosphodiesterase

Saccharomyces cerevisiae Dbr1 is a 405-amino acid RNA debranching enzyme that cleaves the 2′-5′ phosphodiester bonds of the lariat introns formed during pre-mRNA splicing. Debranching appears to be a rate-limiting step for the turnover of intronic RNA, insofar as the steady-state levels of lariat introns are greatly increased in a Δdbr1 strain. To gain insight to the requirements for yeast Dbr1 function, we performed a mutational analysis of 28 amino acids that are conserved in Dbr1 homologs from other organisms. We identified 13 residues (His13, Asp40, Arg45, Asp49, Tyr68, Tyr69, Asn85, His86, Glu87, His179, Asp180, His231 and His233) at which alanine substitutions resulted in lariat intron accumulation in vivo. Conservative replacements at these positions were introduced to illuminate structure–activity relationships. Residues important for Dbr1 function include putative counterparts of the amino acids that comprise the active site of the metallophosphoesterase superfamily, exemplified by the DNA phosphodiesterase Mre11. Using natural lariat RNAs and synthetic branched RNAs as substrates, we found that mutation of Asp40, Asn85, His86, His179, His231 or His233 to alanine abolishes or greatly diminishes debranching activity in vitro. Dbr1 sediments as a monomer and requires manganese as the metal cofactor for debranching.     

NAR Breakthrough Article: Structural basis of lariat RNA recognition by the intron debranching enzyme Dbr1

The enzymatic processing of cellular RNA molecules requires selective recognition of unique chemical and topological features. The unusual 2′,5′-phosphodiester linkages in RNA lariats produced by the spliceosome must be hydrolyzed by the intron debranching enzyme (Dbr1) before they can be metabolized or processed into essential cellular factors, such as snoRNA and miRNA. Dbr1 is also involved in the propagation of retrotransposons and retroviruses, although the precise role played by the enzyme in these processes is poorly understood. Here, we report the first structures of Dbr1 alone and in complex with several synthetic RNA compounds that mimic the branchpoint in lariat RNA. The structures, together with functional data on Dbr1 variants, reveal the molecular basis for 2′,5′-phosphodiester recognition and explain why the enzyme lacks activity toward 3′,5′-phosphodiester linkages. The findings illuminate structure/function relationships in a unique enzyme that is central to eukaryotic RNA metabolism and set the stage for the rational design of inhibitors that may represent novel therapeutic agents to treat retroviral infections and neurodegenerative disease.     

The pattern of amplification and differentiation of Ty1-copia and Ty3-gypsy retrotransposons in Brassicaceae species

One of the causes of genome size expansion is considered to be amplification of retrotransposons. We determined nucleotide sequences of 24 PCR products for each of six retrotransposons in Brassica rapa and Brassica oleracea. Phylogenetic trees of these sequences showed species-specific clades. We also sequenced STF7a homologs and Tto1 homologs, 24 PCR products each, in nine diploids and three allopolyploids, and constructed phylogenetic trees. In these phylogenetic trees, species-specific clades of diploid species were also formed, but retrotransposons of allopolyploids were clustered into the clades of their original genomes, indicating that these two retrotransposons amplified after speciation of the nine diploids. Genetic variation in these retrotransposons may have arisen before emergence of allopolyploid species. There was a positive correlation between the genome size and the average number of substitutions of STF7a and Tto1 homologs in at least seven diploids. The implications of these results in the genome evolution of Brassicaceae are herein discussed.     

Isolation and characterization of the gene encoding yeast debranching enzyme.

Using a genetic screen aimed at identifying cellular factors involved in Ty1 transposition, we have identified a mutation in a host gene that reduces Ty1 transposition frequency. The mutant, dbr1, is also defective in the process of intron turnover. In dbr1 cells, excised introns derived from a variety of pre-mRNAs are remarkably stable and accumulate to levels exceeding that of the corresponding mRNA. The stable excised introns accumulate in the form of a lariat that is missing the linear sequences 3' of the branchpoint. The DBR1 gene has been isolated by complementation of the transposition phenotype. DBR1 is shown to encode debranching enzyme, an RNA processing activity that hydrolyzes the 2'-5' phosphodiester linkage at the branchpoint of excised intron lariats. In Saccharomyces cerevisiae, debranching enzyme plays a requisite role in the rapid turnover of excised introns, yet its function is not essential for viability.     

The p1 protein of the yeast transposon Ty1 can be used for the construction of bi-functional virus-like particles

Virus-like particles (VLPs) containing heterologous proteins are often used as vaccines. Two approaches for the construction of bi-functional VLPs using hybrid protein pl-380 of the TY1 transposon of Saccharomyces yeast are described. We have shown that both C- and N-termini of p1-380 can be used for the expression of heterologous peptides. Peptides from A. Fumigatus Asp f 2, expressed at the C- and/or N-termini of p1-380, did not interfere with VLP self-assembling, were accessible for antibodies and hence were exposed at the VLP surface. Another way to obtain bivalent VLPs is the formation of mixed particles, which co-express two hybrid pl proteins with different heterologous protein fragments at the C-terminus. To do it the yeast cells were transfected with a mixture of two recombinant DNA coding Asp f 2 peptide and green fluorescent protein (Gfp). We have shown that both Asp f 2 peptide and Gfp are expressed within the same particle. To evaluate biological activity of bi-functional VLP a construction containing peptides representing dominant T- and B-cell epitopes of Asp f 2 was produced. Bi-functional particles were more potent in stimulating memory immune responses. These results demonstrate new possibilities of pl-380 based expression system to produce multifunctional VLPs.     

The RNA catabolic enzymes Rex4p, Rnt1p, and Dbr1p show genetic interaction with trans-acting factors involved in processing of ITS1 in Saccharomyces cerevisiae pre-rRNA

Eukaryotes have two types of ribosomes containing either 5.8SL or 5.8SS rRNA that are produced by alternative pre-rRNA processing. The exact processing pathway for the minor 5.8SL rRNA species is poorly documented. We have previously shown that the trans-acting factor Rrp5p and the RNA exonuclease Rex4p genetically interact to influence the ratio between the two forms of 5.8S rRNA in the yeast Saccharomyces cerevisiae. Here we report a further analysis of ITS1 processing in various yeast mutants that reveals genetic interactions between, on the one hand, Rrp5p and RNase MRP, the endonuclease required for 5.8SS rRNA synthesis, and, on the other, Rex4p, the RNase III homolog Rnt1p, and the debranching enzyme Dbr1p. Yeast cells carrying a temperature-sensitive mutation in RNase MRP (rrp2-1) exhibit a pre-rRNA processing phenotype very similar to that of the previously studied rrp5-33 mutant: ITS2 processing precedes ITS1 processing, 5.8SL rRNA becomes the major species, and ITS1 is processed at the recently reported novel site A4 located midway between sites A2 and A3. As in the rrp5-Delta3 mutant, all of these phenotypical processing features disappear upon inactivation of the REX4 gene. Moreover, inactivation of the DBR1 gene in rrp2-1, or the RNT1 gene in rrp5-Delta3 mutant cells also negates the effects of the original mutation on pre-rRNA processing. These data link a total of three RNA catabolic enzymes, Rex4p, Rnt1p, and Dbr1p, to ITS1 processing and the relative production of 5.8SS and 5.8SL rRNA. A possible model for the indirect involvement of the three enzymes in yeast pre-rRNA processing is discussed.     

Ty1-copia group retrotransposons and the evolution of retroelements in the eukaryotes

Ty1-copia group retrotransposons are among the best studied transposable elements in the eukaryotes. This review discusses the extent of these transposons in the eukaryote kingdoms and compares models for the evolution of these genetic elements in the light of recent phylogenetic data. These data show that the Ty1-copia group is widespread among invertebrate eukaryotes, especially in the higher plant kingdom, where these genetic elements are unusually common and heterogeneous in their sequence. The phylogenetic data also suggest that the present day spectrum of Ty1-copia group retrotransposons has been influenced both by divergence during vertical transmission down evolving lineages and by horizontal transmission between distantly related species. Lastly, the factors affecting Ty1-copia group retrotransposon copy number and sequence heterogeneity in eukaryotic genomes and the effects of transpositional quiescence and defective retrotransposons upon evolution of Ty1-copia group retrotransposons are discussed.     

Branchpoint sugar stereochemistry determines the hydrolytic susceptibility of branched RNA fragments by the yeast debranching enzyme (YDBR)

A series of branched RNAs (Y-shaped) related to yeast pre-mRNA splicing intermediates were synthesized incorporating both natural (i.e., ribose) and non-natural (i.e., arabinose, xylose and acyclic nucleoside) branchpoints in order to examine the effect of sugar conformation and phosphodiester configuration on yDBR hydrolytic efficiency. The results indicate that 2'-phosphodiester scission with yDBR occurs only with a cis-arrangement of phosphate groups at the branchpoint (i.e., ribose) thereby discriminating between all other configurations.     

Cloning, expression, and complementation test of the RNA lariat debranching enzyme cDNA from mouse

The RNA lariat debranching enzyme of mouse (mDBR1) was cloned by screening a NIH/3T3 cDNA library. The sequence of full-length mDBR1 cDNA contained a single 515 amino acid open reading frame of 58 kDa protein. Comparison of the amino acid sequence of mDBR1 to other DBR proteins showed 40%, 44%, 43%, 42%, and 80% identity to Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, and human debranching enzymes, respectively. The mDBR1 cDNA was shown to be functional in an interspecies specific complementation experiment, and an in vitro debranching enzyme assay. Mouse DBR1 could complement the intron accumulation phenotype of a S. cerevisiae dbrl null mutant strain. However, the level of complementation depended on the copy number of the mDBR1 cDNA. The integration of the mDBR1 cDNA in the chromosome of S. pombe also complemented both intron accumulation and slow growth phenotypes of the S. pombe dbr1 knock-out mutant strain.     

Gypsy/Ty3-class retrotransposons integrated in the DNA of herring,tunicate, and echinoderms

Eight new examples of retrotransposons of the Gypsy/Ty3 class have been identified in marine species. A 525-nt pol gene-coding region was amplified using degenerate primers from highly conserved regions and has extended the range of recognition of Gypsy/Ty3 far beyond those previously known. The following matrix shows the percentage AA divergence of the translations of this segment of the pol gene coding region. Spr2 Strongylocentrotus purpuratus, sea urchin 39 Por2 Pisaster ochraceus, starfish 46 45 Cprl Clupea pallasi, herring 51 52 41 Cirl Ciona intestinalis, tunicate bar52 49 49 55 P. orchraceus, starfish 55 60 60 62 62 Spr3 S. purpuratus, sea urchin 55 61 60 63 61 24 Tgrl^* Tripneustes gratilla, sea urchin 56 61 60 63 58 26 27 Lvrl^* Lytechinus variegatus, sea urchin 57 62 60 64 62 27 10 29 Sprl^* S. purpuratus 58 61 62 65 61 15 27 30 31 Spr4 S. purpuratus 72 72 74 75 72 73 72 72 73 72 Por3 P. ochraceus The underlines separate three groups of retrotransposons that can be recognized on the basis of this amino acid sequence. The new upper group shows surprising amino acid sequence similarity among members from the DNA of herring, sea urchin, starfish, and a tunicate. For example, the herring element differs by only 41 % from the Ciona element and 46% from the sea urchin element. The group between the lines includes members close to previously known elements (marked by asterisks) and has so far been found only in sea urchins. The two upper groups differ from each other by 55–60% and yet members of both groups (e.g., Sprl and Spr2) are integrated into the DNA of one species-S. purpuratus. Below the lower underline is listed the only known representative of a very distant group, which occurs in starfish DNA. In spite of large divergence, amino acid sequence comparisons indicate that all of the elements shown in the array are members of the LTR-containing class of retrotransposons that includes Gypsy of Drosophila and Ty3 of yeast. Of all known mobile elements this class shows the closest sequence similarity to retroviruses and has the same arrangement of genes as simpler retroviruses.Correspondence to: R.J. Britten     

Human RNA lariat debranching enzyme cDNA complements the phenotypes of Saccharomyces cerevisiae dbr1 and Schizosaccharomyces pombe dbr1 mutants

The cDNA encoding the human RNA lariat debranching enzyme (hDBR1) was identified and cloned by searching the Expressed Sequence Tag (EST) database and screening a HeLa cDNA library, based on predicted amino acid sequence homologies with the Saccharomyces cerevisiae, Schizosaccharomyces pombe and Caenorhabditis elegans debranching enzymes. The hDBR1 cDNA expressed in Escherichia coli showed debranching activity in vitro and was also shown to be functional in an interspecies specific complementation experiment. hDBR1 cDNA in a S.cerevisiae expression vector complemented the intron accumulation phenotype of a S.cerevisiae dbr1 null mutant. Integration of the cDNA for hDBR1 into the ura4 locus of S.pombe also complemented both the intron accumulation and slow growth phenotypes of a S.pombe dbr1 null mutant strain. Comparison of the amino acid sequence of hDBR1 with the other DBR protein sequences showed several conserved regions, with 40, 44 and 43% identity to the S.cerevisiae, S.pombe and C.elegans debranching enzymes, respectively.