Mensur Dlakić

Ssf1p Prevents Premature Processing of an Early Pre-60S Ribosomal Particle

Ssf1p and Ssf2p are two nearly identical and functionally redundant nucleolar proteins. In the absence of Ssf1p and Ssf2p, the 27SA(2) pre-rRNA was prematurely cleaved, inhibiting synthesis of the 27SB and 7S pre-rRNAs and the 5.8S and 25S rRNA components of the large ribosomal subunit. On sucrose gradients, Ssf1p sedimented with pre-60S ribosomal particles. The 27SA(2), 27SA(3), and 27SB pre-rRNAs were copurified with tagged Ssf1p, as were 23 large subunit ribosomal proteins and 21 other proteins implicated in ribosome biogenesis. These included four Brix family proteins, Ssf1p, Rpf1p, Rpf2p, and Brx1p, indicating that the entire family functions in ribosome synthesis. This complex is distinct from recently reported pre-60S complexes in RNA and protein composition. We describe a multistep pathway of 60S preribosome maturation.

Nob1p is required for cleavage of the 3′ end of 18S rRNA

ABSTRACT We report the characterization of a novel factor, Nob1p (Yor056c), which is essential for the synthesis of 40S ribosome subunits. Genetic depletion of Nob1p strongly inhibits the processing of the 20S pre-rRNA to the mature 18S rRNA, leading to the accumulation of high levels of the 20S pre-rRNA together with novel degradation intermediates. 20S processing occurs within a pre-40S particle after its export from the nucleus to the cytoplasm. Consistent with a direct role in this cleavage, Nob1p was shown to be associated with the pre-40S particle and to be present in both the nucleus and the cytoplasm. This suggests that Nob1p accompanies the pre-40S ribosomes during nuclear export. Pre-40S export is not, however, inhibited by depletion of Nob1p.

Prp8, the pivotal protein of the spliceosomal catalytic center, evolved from a retroelement-encoded reverse transcriptase

Prp8 is the largest and most highly conserved protein of the spliceosome, encoded by all sequenced eukaryotic genomes but missing from prokaryotes and viruses. Despite all evidence that Prp8 is an integral part of the spliceosomal catalytic center, much remains to be learned about its molecular functions and evolutionary origin. By analyzing sequence and structure similarities between Prp8 and other protein domains, we show that its N-terminal region contains a putative bromodomain. The central conserved domain of Prp8 is related to the catalytic domain of reverse transcriptases (RTs) and is most similar to homologous enzymes encoded by prokaryotic retroelements. However, putative catalytic residues in this RT domain are only partially conserved and may not be sufficient for the nucleotidyltransferase activity. The RT domain is followed by an uncharacterized sequence region with relatives found in fungal RT-like proteins. This part of Prp8 is predicted to adopt an α-helical structure and may be functionally equivalent to diverse maturase/X domains of retroelements and to the thumb domain of retroviral RTs. Together with a previously identified C-terminal domain that has an RNaseH-like fold, our results suggest evolutionary connections between Prp8 and ancient mobile elements. Prp8 may have evolved by acquiring nucleic acid-binding domains from inactivated retroelements, and their present-day role may be in maintaining proper conformation of the bound RNA cofactors and substrates of the splicing reaction. This is only the second example-the other one being telomerase-of the RT recruitment from a genomic parasite to serve an essential cellular function.

DNA sequence-dependent folding determines the divergence in binding specificities between Maf and other bZIP proteins

Maf family transcription factors are atypical basic region–leucine zipper (bZIP) proteins that contain a variant basic region and an ancillary DNA‐binding region. These proteins recognize extended DNA sequence elements flanking the core recognition element bound by canonical bZIP proteins. We have investigated the causes for the differences in DNA recognition between Maf and other bZIP family proteins through studies of Maf secondary structure, trypsin sensitivity, binding affinity, dissociation rate and DNA contacts. Our results show that specific DNA binding by Maf is coupled to a conformational change involving both the basic and ancillary DNA‐binding regions that depends on the extended DNA sequence elements. Two basic region amino acid residues that differ between Maf and canonical bZIP proteins facilitate the conformational change required for Maf recognition of the extended elements. Nucleotide base contacts made by Maf differ from those made by canonical bZIP proteins. Taken together, our results suggest that the unusual DNA binding specificity of Maf family proteins is mediated by concerted folding of structurally unrelated DNA recognition motifs.

Chromatin silencing protein and pachytene checkpoint regulator Dot1p has a methyltransferase fold

Although protein methylation has been observed for decades, its functional significance has remained largely unclear. Using sensitive profile searches and structural modeling, chromatin silencing protein and meiotic recombination checkpoint regulator Dot1p was identified as a putative protein methyltransferase. Along with recent results that link histone H3 methylation with chromatin silencing, this finding suggests that an expanded combinatorial repertoire of protein modifications affects transcriptional regulation.

A new family of putative insulin receptor-like proteins in C. elegans

I thank Anders Krogh and Gabor Tusnady for providing computer programs for topology prediction of transmembrane proteins. I am grateful to an anonymous referee for useful suggestions. This work was funded by a Special Fellowship from the Leukemia & Lymphoma Society.

A model of the replication fork blocking protein Fob1p based on the catalytic core domain of retroviral integrases

The replication fork blocks are common in both prokaryotes and eukaryotes. In most cases, these blocks are associated with increased levels of mitotic recombination. One of the best‐characterized replication fork blocks in eukaryotes is found in ribosomal DNA (rDNA) repeats of Saccharomyces cerevisiae. It has been shown that the replication fork blocking protein Fob1p regulates the recombination rate and the number of rDNA copies in S. cerevisiae, but the mechanistic aspects of these events are still poorly understood. Sequence profile searches revealed that Fob1p is related to retroviral integrases. Subsequently, the catalytic domain of HIV‐1 integrase was used as a template to build a reliable three‐dimensional model of Fob1p. Structural insights from this study may be useful in explaining Fob1p‐mediated formation of extrachromosomal rDNA circles that accelerate aging in yeast and recombination events that lead to expansion or contraction of rDNA.