The precise DNA arrangement at chromosomal ends and the proteins involved in its maintenance are of crucial importance for genome stability. For the yeast Saccharomyces cerevisiae, this constitutive DNA configuration has remained unknown. We demonstrate here that Gtails of 12-14 bases are present outside of S phase on normal yeast telomeres. Furthermore, the Mre11p protein is essential for the proper establishment of this constitutive end-structure. However, the timing of extended G-tails occurring during S phase is not affected in strains lacking Mre11p. Thus, G-tails are present on yeast chromosomes throughout the cell cycle and the MRX complex is required for their normal establishment. The physical ends of eukaryotic chromosomes, the telomeres, have a very conserved structure and are essential for genome stability (for review, see Blackburn 2001;Chakhparonian and Wellinger 2003). Short, direct DNA repeats constitute the underlying telomeric DNA and the strand running 5Ј to 3Ј toward the end of the chromosomes is usually rich in guanines (the G-rich strand). Lagging-strand synthesis always occurs on this G-rich strand and will leave a short gap at the 5Ј end of the newly synthesized C-rich strand. This gap cannot be filled in by repair, and a 3Ј G-rich overhang, called G-tail, remains. On the other end, leading-strand synthesis is thought to produce a blunt extremity. However, studies of the terminal DNA arrangement in a variety of organisms suggest that a G-tail is a conserved motif for all telomeres (Chakhparonian and Wellinger 2003). Thus, the question arises as to how the blunt-ended DNA ends generated by leading-strand synthesis are converted into ends with a G-tail.Studies in the yeast Saccharomyces cerevisiae have shown that its telomeres acquire detectable G-tails late in S phase, after conventional replication (Wellinger et al. 1993a,b). Moreover, at least on the ends of a linear plasmid, G-tails occur on both, leading-and laggingstrand ends . Surprisingly, these S-phase-specific G-tails can also be detected in cells lacking telomerase, the main activity responsible for replicating telomeric G-strands (Dionne and Wellinger 1996). Collectively, these results suggest that the blunt end left after completion of leading-strand synthesis is processed into an end with a G-tail, presumably by nuclease/helicase activities . Analyses of the requirements to establish a normal telomeric DNA endstructure are hampered by the fact that for wild-type yeast cells, the precise DNA arrangement outside of S phase is unknown.Recent studies on the Mre11p/Rad50p/Xrs2p (MRX) proteins, an evolutionarily conserved complex involved in a number of processes in mitosis and meiosis, revealed that this complex may play a key role in telomere length maintenance in humans, plants, and yeasts (for review, see Haber 1998; D'Amours and Jackson 2002). Yeast cells harboring a deletion of any one of these genes are viable, but display shortened telomeric repeat tracts (Kironmai and Muniyappa 1997; Boulton and Jackson 1998). The Mre11p p...

Two cis-acting RNA trafficking sequences (heterogenous ribonucleoprotein A2 (hnRNP A2)-response elements 1 and 2 or A2RE-1 and A2RE-2) have been identified in HIV-1 vpr and gag mRNAs and were found to confer cytoplasmic RNA trafficking in a murine oligodendrocyte assay. Their activities were assessed during HIV-1 proviral gene expression in COS7 cells. Single point mutations that were shown to severely block RNA trafficking were introduced into each of the A2REs. In both cases, this resulted in a marked decrease in hnRNP A2 binding to HIV-1 genomic RNA in whole cell extracts and hnRNP A2-containing polysomes. This also resulted in an accumulation of HIV-1 genomic RNA in the nucleus and a significant reduction in genomic RNA encapsidation levels. Immunofluorescence analyses revealed altered expression patterns for pr55Gag and particularly that for Vpr. Vpr localization became almost completely nuclear and this was reflected in a significant reduction in virion-associated Vpr levels. These effects coincided with late steps of the viral replication cycle and were not seen at early time points post-transfection. Transcription, splicing, steady state RNA levels, and pr55 Gag processing were not affected. On the other hand, viral replication was markedly compromised in A2RE-2 mutant viruses and this correlated with lowered genomic RNA encapsidation levels. These data reveal new insights into the virus-host interactions between hnRNP A2 and the HIV-1 A2REs and their influence on the patterns of HIV-1 gene expression and viral assembly. Human immunodeficiency virus type 1 (HIV-1)1 is the cause of acquired immunodeficiency syndrome (AIDS). Transcription of the integrated provirus produces one primary 9-kb transcript that is spliced to produce three size classes of RNA (1). The smallest size class, the 2-kb RNAs, is constitutively exported to the cytosol early in the HIV-1 replication cycle and encodes for the regulatory proteins Tat, Rev, and Nef. Late in the replication cycle, the two other size classes of RNA, the unspliced, 9-kb genomic RNA and the singly spliced, 4-kb RNAs make their way to the cytosol due principally to the activity of Rev, which binds to the Rev responsive element (RRE) present in these RNAs (2). Whereas an abundant amount of information is available about the mechanisms, cellular cofactors, and regulation involved in Rev-mediated RNA nucleocytoplasmic transport (3), very little is understood about HIV-1 RNA trafficking following Rev's disengagement in the cytosol. Recent work demonstrates a role for the cellular human Rev-interacting protein (hRIP) at this step (4). The HIV-1 structural protein, pr55Gag also plays a role at this late step by binding to RNA via it N-terminal matrix (MA) and C-terminal nucleocapsid (NC) domains (5-7). pr55Gag association to molecular motor proteins (8) provides a mechanism by which RNA trafficking is achieved within the cytoplasm. In support of the existence for a trafficking mechanism are data showing that kinesins and microtubules are both necessary for the traffi...

In the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 pre-mRNA, different regions in the introns flanking alternative exon 7B have been implicated in the production of the A1 and A1B mRNA splice isoforms. Among these, the CE1a and CE4 elements, located downstream of common exon 7 and alternative exon 7B, respectively, are bound by hnRNP A1 to promote skipping of exon 7B in vivo and distal 5 splice site selection in vitro. Here, we report that CE1a is flanked by an additional high affinity A1 binding site (CE1d). In a manner similar to CE1a, CE1d affects 5 splice site selection in vitro. Consistent with a role for hnRNP A1 in the activity of CE1d, a mutation that abrogates A1 binding abolishes distal 5 splice site activation. Moreover, the ability of CE1d to stimulate distal 5 splice site usage is lost in an HeLa extract depleted of hnRNP A/B proteins, and the addition of recombinant A1 restores the activity of CE1d. Notably, distal 5 splice site selection mediated by A1 binding sites is not compromised in an extract prepared from mouse cells that are severely deficient in hnRNP A1 proteins. In this case, we show that hnRNP A2 compensates for the A1 deficiency. Further studies with the CE4 element reveal that it also consists of two distinct portions (CE4m and CE4p), each one capable of promoting distal 5 splice site use in an hnRNP A1-dependent manner. The presence of multiple A1/A2 binding sites downstream of common exon 7 and alternative exon 7B probably plays an important role in maximizing the activity of hnRNP A1/A2 proteins.The alternative splicing of mRNA precursors (pre-mRNAs) 1 is a major contributor to the diversity of the mammalian proteome (1-3). The control of splice site selection therefore has profound implications in the production of protein isoforms with different functions. Recent progress in uncovering the molecular strategies that control alternative splicing has led to the identification of many types of sequence elements that influence either positively or negatively the selection of the alternative splice sites. Exonic splicing enhancers are bound by specific members of the SR protein family that can enforce the use of weak 5Ј and 3Ј splice sites (reviewed in Ref. 4). Enhancer elements have also been described in the introns flanking some alternative exons (5-9). Other types of proteins, including members of the hnRNP F/H family of proteins, can bind specifically to intron or exon control elements and hence can contribute to enhancer activity (10 -14).Elements that reduce the use of a neighboring splice site are also important in the control of splice site selection. In many cases, the activity of splicing silencers can be mediated by proteins that inhibit specific steps of splice site recognition or spliceosome assembly. A frequent example of this kind of splicing control in mammals involves the polypyrimidine tract-binding protein, which binds to some 3Ј splice sequences and prevents U2AF binding (15)(16)(17)(18)(19). Other examples uncovered in mammalian pre-mRNAs include the bindi...

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Telomeres are essential features of linear genomes that are crucial for chromosome stability. Telomeric DNA is usually replenished by telomerase. Deletion of genes encoding telomerase components leads to telomere attrition with each cycle of DNA replication, eventually causing cell senescence or death. In the Saccharomyces cerevisiae strain W303, telomerase-null populations bypass senescence and, unless EXO1 is also deleted, this survival is RAD52 dependent. Unexpectedly, we found that the S. cerevisiae strain S288C could survive the removal of RAD52 and telomerase at a low frequency without additional gene deletions. These RAD52-independent survivors were propagated stably and exhibited a telomere organization typical of recombination between telomeric DNA tracts, and in diploids behaved as a multigenic trait. The polymerase-d subunit Pol32 was dispensable for the maintenance of RAD52-independent survivors. The incidence of this rare escape was not affected by deletion of other genes necessary for RAD52-dependent survival, but correlated with initial telomere length. If W303 strains lacking telomerase and RAD52 first underwent telomere elongation, rare colonies could then bypass senescence. We suggest that longer telomeres provide a more proficient substrate for a novel telomere maintenance mechanism that does not rely on telomerase, RAD52, or POL32.

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BackgroundThe first telomerase-associated protein (Est1) was isolated in yeast due to its essential role in telomere maintenance. The human counterparts EST1A, EST1B, and EST1C perform diverse functions in nonsense-mediated mRNA decay (NMD), telomere length homeostasis, and telomere transcription. Although Est1 and EST1A/B interact with the catalytic subunit of yeast and human telomerase (Est2 and TERT, respectively), the molecular determinants of these interactions have not been elaborated fully.ResultsTo investigate the functional conservation of the EST1 protein family, we performed protein-protein interaction mapping and structure-function analysis. The domain in hEST1A most conserved between species, containing a TPR (tricotetrapeptide repeat), was sufficient for interaction of hEST1A with multiple fragments of hTERT including the N-terminus. Two mutations within the hTERT N-terminus that perturb in vivo function (NAAIRS92, NAAIRS122) did not affect this protein interaction. ScEst1 hybrids containing the TPR of hEST1A, hEST1B, or hEST1C were expressed in yeast strains lacking EST1, yet they failed to complement senescence. Point mutations within and outside the cognate ScEst1 TPR, chosen to disrupt a putative protein interaction surface, resulted in telomere lengthening or shortening without affecting recruitment to telomeres.ConclusionsThese results identify a domain encompassing the TPR of hEST1A as an hTERT interaction module. The TPR of S. cerevisiae Est1 is required for telomerase-mediated telomere length maintenance in a manner that appears separable from telomere recruitment. Discrete residues in or adjacent to the TPR of Est1 also regulate telomere length homeostasis.

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