Sheila Lutz

Multiple fates of L1 retrotransposition intermediates in cultured human cells

ABSTRACT LINE-1 (L1) retrotransposons comprise ∼17% of human DNA, yet little is known about L1 integration. Here, we characterized 100 retrotransposition events in HeLa cells and show that distinct DNA repair pathways can resolve L1 cDNA retrotransposition intermediates. L1 cDNA resolution can lead to various forms of genetic instability including the generation of chimeric L1s, intrachromosomal deletions, intrachromosomal duplications, and intra-L1 rearrangements as well as a possible interchromosomal translocation. The L1 retrotransposition machinery also can mobilize U6 snRNA to new genomic locations, increasing the repertoire of noncoding RNAs that are mobilized by L1s. Finally, we have determined that the L1 reverse transcriptase can faithfully replicate its own transcript and has a base misincorporation error rate of ∼1/7,000 bases. These data indicate that L1 retrotransposition in transformed human cells can lead to a variety of genomic rearrangements and suggest that host processes act to restrict L1 integration in cultured human cells. Indeed, the initial steps in L1 retrotransposition may define a host/parasite battleground that serves to limit the number of active L1s in the genome.

Sus1, Cdc31, and the Sac3 CID region form a conserved interaction platform that promotes nuclear pore association and mRNA export.

Summary The yeast Sac3:Cdc31:Sus1:Thp1 (TREX-2) complex facilitates the repositioning and association of actively transcribing genes with nuclear pores (NPCs)—“gene gating”—that is central to integrating transcription, processing, and mRNA nuclear export. We present here the crystal structure of Sus1 and Cdc31 bound to a central region of Sac3 (the CID domain) that is crucial for its function. Sac3CID forms a long, gently undulating α helix around which one Cdc31 and two Sus1 chains are wrapped. Sus1 has an articulated helical hairpin fold that facilitates its wrapping around Sac3. In vivo studies using engineered mutations that selectively disrupted binding of individual chains to Sac3 indicated that Sus1 and Cdc31 function synergistically to promote NPC association of TREX-2 and mRNA nuclear export. These data indicate Sac3CID provides a scaffold within TREX-2 to integrate interactions between protein complexes to facilitate the coupling of transcription and mRNA export during gene expression.

Functional and structural characterization of the mammalian TREX-2 complex that links transcription with nuclear messenger RNA export

Export of messenger RNA (mRNA) from the nucleus to the cytoplasm is a critical step in the gene expression pathway of eukaryotic cells. Here, we report the functional and structural characterization of the mammalian TREX-2 complex and show how it links transcription/processing with nuclear mRNA export. Mammalian TREX-2 is based on a germinal-centre associated nuclear protein (GANP) scaffold to which ENY2, PCID2 and centrins bind and depletion of any of these components inhibits mRNA export. The crystal structure of the GANP:ENY2 complex shows that two ENY2 chains interact directly with GANP, but they have different orientations from those observed on yeast Sac3. GANP is required to recruit ENY2 to nuclear pore complexes (NPCs), but ENY2 is not necessary to recruit GANP, which requires both its CID and MCM3AP domains, together with nucleoporin Nup153. GANP and ENY2 associate with RNA polymerase II and inhibition of mRNA processing redistributes GANP from NPCs into nuclear foci indicating that mammalian TREX-2 is associated with transcription. Thus, we implicate TREX-2 as an integral component of the mammalian mRNA export machinery where it links transcription and nuclear export by facilitating the transfer of mature mRNPs from the nuclear interior to NPCs.

Allelic heterogeneity in LINE-1 retrotransposition activity

De novo LINE-1 (long interspersed element-1, or L1) retrotransposition events are responsible for approximately 1/1,000 disease-causing mutations in humans. Previously, L1.2 was identified as the likely progenitor of a mutagenic insertion in the factor VIII gene in a patient with hemophilia A. It subsequently was shown to be one of a small number of active L1s in the human genome. Here, we demonstrate that L1.2 is present at an intermediate insertion allele frequency in worldwide human populations and that common alleles (L1.2A and L1.2B) exhibit an approximately 16-fold difference in their ability to retrotranspose in cultured human HeLa cells. Chimera analysis revealed that two amino acid substitutions (S1259L and I1220M) downstream of the conserved cysteine-rich motif in L1 open reading frame 2 are largely responsible for the observed reduction in L1.2A retrotransposition efficiency. Thus, common L1 alleles can vary widely in their retrotransposition potential. We propose that such allelic heterogeneity can influence the potential L1 mutational load present in an individual genome.

A maize acetyl-coenzyme A carboxylase cDNA sequence.

ACCase (EC 6.4.1.2) catalyzes synthesis of the malonylCOA required for subsequent synthesis of fatty acids and secondary metabolites in plants. ACCase activity is positively correlated with rates of fatty acid synthesis in both leaves and developing oil seeds, and it is likely to play a key regulatory role in plant lipid synthesis (Post-Beittenmiller et al., 1993). In maize (Zeu muys), most ACCase activity is associated with a high molecular weight, dimeric, MF, plastid-localized polypeptide that is sensitive to inhibition by aryloxyphenoxypropionate and cyclohexanedione herbicides (Egli et al., 1993). Complete coding sequences for MF ACCase polypeptides have been published for wheat (Gornicki et al., 1994) and for severa1 dicotyledons (Roesler et al., 1994; Schulte et al., 1994; Shorrosh e t al., 1994). Part ia1 m a i z e and rice ACCase cDNAs have also been reported (Ashton et al., 1994; T. Sasaki, unpublished data). Here we describe the complete coding sequence of a maize ACCase (Table I). Antiserum to the major maize ACCase polypeptide (ACCase I; Egli et al., 1993) was used to select potential maize ACCase cDNA clones from an oligo(dT)-primed Agtll expression library derived from A188 maize seedling leaf (Dr. Stephen Gantt, University of Minnesota). Plaques from 14 of 800,000 clones were strongly recognized by the antiserum. Four clones that contained 3.5to 5.4-kb inserts were partially sequenced and found to be identical. The remaining 5’ coding sequence was obtained by partia1 sequencing of a 15-kb genomic clone whose 3‘ end hybridized to an ACCase cDNA probe (nt 3900-5932). The corresponding cDNA sequence was obtained by three successive rounds of RT-PCR, using oligonucleotide primers based on genomic apparent exon (5’) and known cDNA (3’) sequences. PCR products corresponded to nt 1 to 240, 217 to 610, and 537 to 2094 of the final sequence and were cloned into PCR-script (Stratagene). The original 5.4-kb cDNA clone No. 18-5 and PCR products from at least three

Mutational Uncoupling of the Role of Sus1 in Nuclear Pore Complex Targeting of an mRNA Export Complex and Histone H2B Deubiquitination

Sus1 is an evolutionary conserved protein that functions both in transcription and mRNA export and has been proposed to contribute to coupling these processes in yeast. Sus1 mediates its different roles as a component of both the histone H2B deubiquitinating module (Sus1-Sgf11-Ubp8-Sgf73) of the SAGA (Spt-Ada-Gcn5 acetyltransferase) transcriptional co-activator and the mRNA export complex, TREX-2 (Sus1-Sac3-Thp1-Cdc31). We have dissected the different functions of Sus1 with respect to its partitioning in transcription and export complexes using a mutational approach. Here we show that the sus1–10 (E18A, S19A, and G20A) and sus1–12 (V73A and D75A) alleles of Sus1 can be dissociated from TREX-2 while leaving its interaction with SAGA largely intact. Conversely, the binding to both TREX-2 and SAGA was impaired in the sus1–11 allele (G37A and W38A), in which two highly conserved residues were mutated. In vitro experiments demonstrated that dissociation of mutant Sus1 from its partners is caused by a reduced affinity toward the TREX-2 subunit, Sac3, and the SAGA factor, Sgf11, respectively. Consistent with the biochemical data, these sus1 mutant alleles showed differential genetic relationships with SAGA and mRNA export mutants. In vivo, all three sus1 mutants were impaired in targeting TREX-2 (i.e. Sac3) to the nuclear pore complexes and exhibited nuclear mRNA export defects. This study has implications for how Sus1, in combination with distinct interaction partners, can regulate diverse aspects of gene expression.

RNA-RNA interactions and pre-mRNA mislocalization as drivers of group II intron loss from nuclear genomes

Significance For over three decades, group II introns have been conjectured to be the ancestors of splicesomal introns, but there are no group II introns in extant nuclear genomes. Might these introns have been expunged as spliceosomal introns proliferated? We showed previously that nuclear expression of a group II intron in yeast resulted specifically in down-regulation of its host gene. Here, we report on the discovery that pre-mRNA mislocalization and a consequent interaction between the pre-mRNA or intron and spliced mRNA together account for the mechanism of gene silencing. Our data support the hypothesis that such roadblocks to gene expression resulted in purging of group II introns from nuclear genomes while promoting the evolution of spliceosomal introns. Group II introns are commonly believed to be the progenitors of spliceosomal introns, but they are notably absent from nuclear genomes. Barriers to group II intron function in nuclear genomes therefore beg examination. A previous study showed that nuclear expression of a group II intron in yeast results in nonsense-mediated decay and translational repression of mRNA, and that these roadblocks to expression are group II intron-specific. To determine the molecular basis for repression of gene expression, we investigated cellular dynamics of processed group II intron RNAs, from transcription to cellular localization. Our data show pre-mRNA mislocalization to the cytoplasm, where the RNAs are targeted to foci. Furthermore, tenacious mRNA–pre-mRNA interactions, based on intron-exon binding sequences, result in reduced abundance of spliced mRNAs. Nuclear retention of pre-mRNA prevents this interaction and relieves these expression blocks. In addition to providing a mechanistic rationale for group II intron-specific repression, our data support the hypothesis that RNA silencing of the host gene contributed to expulsion of group II introns from nuclear genomes and drove the evolution of spliceosomal introns.

Co-translational Localization of an LTR-Retrotransposon RNA to the Endoplasmic Reticulum Nucleates Virus-Like Particle Assembly Sites

The transcript of retrovirus-like transposons functions as an mRNA for synthesis of capsid and replication proteins and as the genomic RNA of virus-like particles (VLPs), wherein the genome is replicated. Retrotransposon RNA and proteins coalesce in a cytoplasmic focus, or retrosome, to initiate VLP assembly, but it is not known how the retrosome is nucleated. We determined how the RNA and Gag protein of the Saccharomyces cerevisiae Ty1 retrotransposon are directed to the retrosome. We found that Ty1 RNA is translated in association with signal recognition particle (SRP), a universally conserved chaperone that binds specific ribosome-nascent chain (RNC) complexes and targets the nascent peptide to the endoplasmic reticulum (ER). Gag is translocated to the ER lumen; yet, it is also found in the cytoplasm, associated with SRP-RNC complexes. In the absence of ER translocation, Gag is synthesized but rapidly degraded, and Ty1 RNA does not coalesce in retrosomes. These findings suggest that Gag adopts a stable conformation in the ER lumen, is retrotranslocated to the cytoplasm, binds to Ty1 RNA on SRP-RNC complexes and multimerizes to nucleate retrosomes. Consistent with this model, we show that slowing the rate of co-translational ER translocation by limiting SRP increases the prevalence of retrosomes, while suppressing the translocation defect of srp hypomorphs by slowing translational elongation rapidly decreases retrosome formation. Thus, retrosomes are dynamic foci of Ty1 RNA-RNC complexes whose formation is modulated by the rate of co-translational ER translocation. Together, these findings suggest that translating Ty1 mRNA and the genomic RNA of VLPs originate in a single pool and moreover, that co-translational localization of Ty1 RNA nucleates the presumptive VLP assembly site. The separation of nascent Gag from its RNA template by transit through the ER allows Gag to bind translating Ty1 RNA without displaying a cis-preference for its encoding RNA.

Paralog-Specific Functions of RPL7A and RPL7B Mediated by Ribosomal Protein or snoRNA Dosage in Saccharomyces cerevisiae.

Most ribosomal proteins in Saccharomyces cerevisiae are encoded by two paralogs that additively produce the optimal protein level for cell growth. Nonetheless, deleting one paralog of most ribosomal protein gene pairs results in a variety of phenotypes not observed when the other paralog is deleted. To determine whether paralog-specific phenotypes associated with deleting RPL7A or RPL7B stem from distinct functions or different levels of the encoded isoforms, the coding region and introns of one paralog, including an intron-embedded snoRNA (small nucleolar RNA) gene, were exchanged with that of the other paralog. Among mutants harboring a single native or chimeric RPL7 allele, expression from the RPL7A locus exceeded that from the RPL7B locus, and more Rpl7a was expressed from either locus than Rpl7b. Phenotypic differences in tunicamycin sensitivity, ASH1 mRNA localization, and mobility of the Ty1 retrotransposon were strongly correlated with Rpl7 and ribosome levels, but not with the Rpl7 or snoRNA isoform expressed. Although Ty1 RNA is cotranslationally localized, depletion of Rpl7 minimally affected synthesis of Ty1 Gag protein, but strongly influenced Ty1 RNA localization. Unlike the other processes studied, Ty1 cDNA accumulation was influenced by both the level and isoform of Rpl7 or snoRNA expressed. These cellular processes had different minimal threshold values for Rpl7 and ribosome levels, but all were functional when isoforms of either paralog were expressed from the RPL7A locus or both RPL7 loci. This study illustrates the broad range of phenotypes that can result from depleting ribosomes to different levels.