Faculty Bibliography

Long interspersed nuclear elements (LINEs or L1s) comprise approximately 17% of human DNA; however, only about 60 of the approximately 400,000 L1s are mobile. Using a retrotransposition assay in cultured human cells, we demonstrate that L1-encoded proteins predominantly mobilize the RNA that encodes them. At much lower levels, L1-encoded proteins can act in trans to promote retrotransposition of mutant L1s and other cellular mRNAs, creating processed pseudogenes. Mutant L1 RNAs are mobilized at 0.2 to 0.9% of the retrotransposition frequency of wild-type L1s, whereas cellular RNAs are mobilized at much lower frequencies (ca. 0.01 to 0.05% of wild-type levels). Thus, we conclude that L1-encoded proteins demonstrate a profound cis preference for their encoding RNA. This mechanism could enable L1 to remain retrotransposition competent in the presence of the overwhelming number of nonfunctional L1s present in human DNA.

The Saccharomyces cerevisiae genome database contains two ORFs with homology to aquaporins, AQY1 and AQY2. Aqy1p has been shown to be a functional aquaporin in some strains, such as Sigma1278b. AQY2 is disrupted by a stop codon in most strains; however, Sigma1278b has an intact ORF. Because Sigma1278b Aqy2p has an intracellular localization in Xenopus oocytes and in yeast, other strains of yeast were examined. Aqy2p from Saccharomyces chevalieri has a single amino acid in the third transmembrane domain (Ser-141) that differs from Sigma1278b Aqy2p (Pro-141). S. chevalieri Aqy2p is a functional water channel in oocytes and traffics to the plasma membrane of yeast. The Sigma1278b parental strain, the aqy1-aqy2 double null yeast, and null yeast expressing S. chevalieri Aqy2p were examined under various conditions. Comparison of these strains revealed that the aquaporin null cells were more aggregated and their surface was more hydrophobic. As a result, the aquaporin null cells were more flocculent and more efficient at haploid invasive growth. Despite its primary intracellular localization, Sigma1278b Aqy2p plays a role in yeast similar to Aqy1p and S. chevalieri Aqy2p. In addition, Aqy1p and Aqy2p can affect cell surface properties and may provide an advantage by dispersing the cells during starvation or during sexual reproduction.

L1 elements are human transposons which replicate via an RNA intermediate. At least 15% of the human genome is composed of L1 sequence. An important initial step in the transposition reaction is nicking of the genomic DNA by L1 endonuclease (L1 EN). In vivo much of the genome exists in the form of chromatin or is undergoing biochemical transactions such as transcription, replication or repair, which may alter the accessibility of the L1 transposition machinery to DNA. To investigate this possibility we have examined the effect of substrate chromatinization on the ability of L1 EN to nick DNA. We find that DNA incorporated into nucleosomes is generally refractory to nicking by L1 EN. Interestingly, nicking of a minority of DNA sequences is enhanced when included in chromatin. Thus, dynamic epigenetic factors such as chromatinization are likely to influence the relatively permanent placement of L1 and other retroelements in the human genome.

The retroviral protease is a key enzyme in a viral multienzyme complex that initiates an ordered sequence of events leading to virus assembly and propagation. Viral peptides are initially synthesized as polyprotein precursors; these precursors undergo a number of proteolytic cleavages executed by the protease in a specific and presumably ordered manner. To determine the role of individual protease cleavage sites in Ty1, a retrotransposon from Saccharomyces cerevisiae, the cleavage sites were systematically mutagenized. Altering the cleavage sites of the yeast Ty1 retrotransposon produces mutants with distinct retrotransposition phenotypes. Blocking the Gag/PR site also blocks cleavage at the other two cleavage sites, PR/IN and IN/RT. In contrast, mutational block of the PR/IN or IN/RT sites does not prevent cleavage at the other two sites. Retrotransposons with mutations in each of these sites have transposition defects. Mutations in the PR/IN and IN/RT sites, but not in the Gag/PR site, can be complemented in trans by endogenous Ty1 copies. Hence, the digestion of the Gag/PR site and release of the protease N terminus is a prerequisite for processing at the remaining sites; cleavage of PR/IN is not required for the cleavage of IN/RT, and vice versa. Of the three cleavage sites in the Gag-Pol precursor, the Gag/PR site is processed first. Thus, Ty1 Gag-Pol processing proceeds by an ordered pathway.

Active nuclear import of the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC) is essential for the productive infection of nondividing cells. Nuclear import of the PIC is mediated by the HIV-1 matrix protein, which also plays several critical roles during viral entry and possibly during virion production facilitating the export of Pr55(Gag) and genomic RNA. Using a yeast two-hybrid screen, we identified a novel human virion-associated matrix-interacting protein (VAN) that is highly conserved in vertebrates and expressed in most human tissues. Its expression is upregulated upon activation of CD4(+) T cells. VAN is efficiently incorporated into HIV-1 virions and, like matrix, shuttles between the nucleus and cytoplasm. Furthermore, overexpression of VAN significantly inhibits HIV-1 replication in tissue culture. We propose that VAN regulates matrix nuclear localization and, by extension, both nuclear import of the PIC and export of Pr55(Gag) and viral genomic RNA during virion production. Our data suggest that this regulatory mechanism reflects a more global process for regulation of nucleocytoplasmic transport.

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.

The yeast Sir2 protein, required for transcriptional silencing, has an NAD(+)-dependent histone deacetylase (HDA) activity. Yeast extracts contain a NAD(+)-dependent HDA activity that is eliminated in a yeast strain from which SIR2 and its four homologs have been deleted. This HDA activity is also displayed by purified yeast Sir2p and homologous Archaeal, eubacterial, and human proteins, and depends completely on NAD(+) in all species tested. The yeast NPT1 gene, encoding an important NAD(+) synthesis enzyme, is required for rDNA and telomeric silencing and contributes to silencing of the HM loci. Null mutants in this gene have significantly reduced intracellular NAD(+) concentrations and have phenotypes similar to sir2 null mutants. Surprisingly, yeast from which all five SIR2 homologs have been deleted have relatively normal bulk histone acetylation levels. The evolutionary conservation of this regulated activity suggests that the Sir2 protein family represents a set of effector proteins in an evolutionarily conserved signal transduction pathway that monitors cellular energy and redox states.

Human L1 retrotransposons can produce DNA transduction events in which unique DNA segments downstream of L1 elements are mobilized as part of aberrant retrotransposition events. That L1s are capable of carrying out such a reaction in tissue culture cells was elegantly demonstrated. Using bioinformatic approaches to analyze the structures of L1 element target site duplications and flanking sequence features, we provide evidence suggesting that approximately 15% of full-length L1 elements bear evidence of flanking DNA segment transduction. Extrapolating these findings to the 600,000 copies of L1 in the genome, we predict that the amount of DNA transduced by L1 represents approximately 1% of the genome, a fraction comparable with that occupied by exons.