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

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.     

A second base pair interaction between U3 small nucleolar RNA and the 5'-ETS region is required for early cleavage of the yeast pre-ribosomal RNA

In eukaryotes, U3 snoRNA is essential for pre-rRNA maturation. Its 5'-domain was found to form base pair interactions with the 18S and 5'-ETS parts of the pre-rRNA. In Xenopus laevis, two segments of U3 snoRNA form base-pair interactions with the 5'-ETS region and only one of them is essential to the maturation process. In Saccharomyces cerevisiae, two similar U3 snoRNA-5' ETS interactions are possible; but, the functional importance of only one of them had been tested. Surprisingly, this interaction, which corresponds to the non-essential one in X. laevis, is essential for cell growth and pre-rRNA maturation in yeast. In parallel with [Dutca et al. (2011) The initial U3 snoRNA:pre-rRNA base pairing interaction required for pre-18S rRNA folding revealed by in vivo chemical probing. Nucleic Acids Research, 39, 5164-5180], here we show, that the second possible 11-bp long interaction between the 5' domain of S. cerevisiae U3 snoRNA and the pre-rRNA 5'-ETS region (helix VI) is also essential for pre-rRNA processing and cell growth. Compensatory mutations in one-half of helix VI fully restored cell growth. Only a partial restoration of growth was obtained upon extension of compensatory mutations to the entire helix VI, suggesting sequence requirement for binding of specific proteins. Accordingly, we got strong evidences for a role of segment VI in the association of proteins Mpp10, Imp4 and Imp3.     

A yeast small nuclear RNA is required for normal processing of pre-ribosomal RNA.

In Saccharomyces cerevisiae, seven snRNAs (snR3, 4, 5, 8, 9, 10 and 17) are retained in the nucleus under conditions in which nucleoplasmic RNAs are lost, and may be nucleolar. All of these snRNAs show properties consistent with hydrogen bonding to pre-ribosomal RNAs; snR5 and 8 with 20S pre-rRNA, snR3, 4, 10 and 17 with 35S pre-rRNA and snR9 with 20-35S RNA. Strains lacking snR10 are impaired in growth and specifically defective in the processing of 35S RNA. Processing is slowed, leading to 35S RNA accumulation and most cleavage occurs, not at the normal sites, but at sites which in wild-type strains are used for subsequent steps in rRNA maturation.     

FLASH is required for the endonucleolytic cleavage of histone pre-mRNAs but is dispensable for the 5' exonucleolytic degradation of the downstream cleavage product

3'-end cleavage of histone pre-mRNAs is catalyzed by CPSF-73 and requires the interaction of two U7 snRNP-associated proteins, FLASH and Lsm11. Here, by using scanning mutagenesis we identify critical residues in human FLASH and Lsm11 that are involved in the interaction between these two proteins. We also demonstrate that mutations in the region of FLASH located between amino acids 50 and 99 do not affect binding of Lsm11. Interestingly, these mutations convert FLASH into an inhibitory protein that reduces in vitro processing efficiency of highly active nuclear extracts. Our results suggest that this region in FLASH in conjunction with Lsm11 is involved in recruiting a yet-unknown processing factor(s) to histone pre-mRNA. Following endonucleolytic cleavage of histone pre-mRNA, the downstream cleavage product (DCP) is degraded by the 5'-3' exonuclease activity of CPSF-73, which also depends on Lsm11. Strikingly, while cleavage of histone pre-mRNA is stimulated by FLASH and inhibited by both dominant negative mutants of FLASH and anti-FLASH antibodies, the 5'-3' degradation of the DCP is not affected. Thus, the recruitment of FLASH to the processing complex plays a critical role in activating the endonuclease mode of CPSF-73 but is dispensable for its 5'-3' exonuclease activity. These results suggest that CPSF-73, the catalytic component in both reactions, can be recruited to histone pre-mRNA largely in a manner independent of FLASH, possibly by a separate domain in Lsm11.     

Base pairing between U3 and the pre-ribosomal RNA is required for 18S rRNA synthesis.

The nucleolus, the site of pre-ribosomal RNA (pre-rRNA) synthesis and processing in eukaryotic cells, contains a number of small nucleolar RNAs (snoRNAs). Yeast U3 snoRNA is required for the processing of 18S rRNA from larger precursors and contains a region complementary to the pre-rRNA. Substitution mutations in the pre-rRNA which disrupt this base pairing potential are lethal and prevent synthesis of 18S rRNA. These mutant pre-rRNAs show defects in processing which closely resemble the effects of genetic depletion of components of the U3 snoRNP. Co-expression of U3 snoRNAs which carry compensatory mutations allows the mutant pre-rRNAs to support viability and synthesize 18S rRNA at high levels. Pre-rRNA processing steps which are blocked by the external transcribed spacer region mutations are largely restored by expression of the compensatory U3 mutants. Pre-rRNA processing therefore requires direct base pairing between snoRNA and the substrate. Base pairing with the substrate is thus a common feature of small RNAs involved in mRNA and rRNA maturation.     

5' exon requirement for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA and identification of a cryptic 5' splice site in the 3' exon

The intervening sequence (IVS) of the Tetrahymena thermophila ribosomal RNA precursor undergoes accurate self-splicing in vitro. The work presented here examines the requirement for Tetrahymena rRNA sequences in the 5' exon for the accuracy and efficiency of splicing. Three plasmids were constructed with nine, four and two nucleotides of the natural 5' exon sequence, followed by the IVS and 26 nucleotides of the Tetrahymena 3' exon. RNA was transcribed from these plasmids in vitro and tested for self-splicing activity. The efficiency of splicing, as measured by the production of ligated exons, is reduced as the natural 5' exon sequence is replaced with plasmid sequences. Accurate splicing persists even when only four nucleotides of the natural 5' exon sequence remain. When only two nucleotides of the natural exon remain, no ligated exons are observed. As the efficiency of the normal reaction diminishes, novel RNA species are produced in increasing amounts. The novel RNA species were examined and found to be products of aberrant reactions of the precursor RNA. Two of these aberrant reactions involve auto-addition of GTP to sites six nucleotides and 52 nucleotides downstream from the 3' splice site. The former site occurs just after the sequence GGU, and may indicate the existence of a GGU-binding site within the IVS RNA. The latter site follows the sequence CUCU, which is identical with the four nucleotides preceding the 5' splice site. This observation led to a model where where the CUCU sequence in the 3' exon acts as a cryptic 5' splice site. The model predicted the existence of a circular RNA containing the first 52 nucleotides of the 3' exon. A small circular RNA was isolated and partially sequenced and found to support the model. So, a cryptic 5' splice site can function even if it is located downstream from the 3' splice site. Precursor RNA labeled at its 5' end, presumably by a GTP exchange reaction mediated by the IVS, is also described.     

The yeast Hansenula wingei U3 snoRNA gene contains an intron and its coding sequence co-evolved with the 5' ETS region of the pre-ribosomal RNA.

The 5' external transcribed spacer (ETS) region of the pre-rRNA in Saccharomyces cerevisiae contains a sequence with 10 bp of perfect complementarity to the U3 snoRNA. Base pairing between these sequences has been shown to be required for 18S rRNA synthesis, although interaction over the full 10 bp of complementarity is not required. We have identified the homologous sequence in the 5' ETS from the evolutionarily distant yeast Hansenula wingei; unexpectedly, this shows two sequence changes in the region predicted to base pair to U3. By PCR amplification and direct RNA sequencing, a single type of U3 snoRNA coding sequence was identified in H. wingei. As in the S. cerevisiae U3 snoRNA genes, it is interrupted by an intron with features characteristic of introns spliced in a spliceosome. Consequently, this unusual property is not restricted to the yeast genus Saccharomyces. The introns of the H. wingei and S. cerevisiae U3 genes show strong differences in length and sequence, but are located at the same position in the U3 sequence, immediately upstream of the phylogenetically conserved Box A region. The 3' domains of the H. wingei and S. cerevisiae U3 snoRNAs diverge strongly in primary sequence, but have very similar predicted secondary structures. The 5' domains, expected to play a direct role in pre-ribosomal RNA maturation, are more conserved. The sequence predicted to base pair to the pre-rRNA contains two nucleotide substitutions in H. wingei that restore 10 bp of perfect complementarity to the 5' ETS. This is a strong phylogenetic evidence for the importance of the U3/pre-rRNA interaction.     

Skp1p regulates Soi3p/Rav1p association with endosomal membranes but is not required for vacuolar ATPase assembly

Skp1p is an essential component of SCF-type E3 ubiquitin ligase complexes and associates with these through binding to F-box proteins. Skp1p also binds F-box proteins in a number of non-SCF complexes. The Skp1p-associated yeast protein Soi3p/Rav1p (hereafter referred to as Rav1p) is a component of the RAVE complex required for regulated assembly of vacuolar ATPase (V-ATPase). Rav1p is also involved in transport of TGN proteins and endocytic cargo between early and late endosomes. To evaluate the role of Skp1p in the RAVE complex, we made use of the fact that overexpression of Rav1p is toxic because it sequesters Skp1p from essential interactions. We isolated a separation of function allele of SKP1, skp1(Asn108Tyr), that completely abrogated the Rav1p interaction but allowed Skp1p to perform other essential cellular functions. Cells containing the skp1(Asn108Tyr) allele as the sole source of Skp1p exhibited normal V-ATPase assembly and activity. However, in the skp1(Asn108Tyr) mutant strain, the membrane-associated pool of Rav1-green fluorescent protein was increased, suggesting that Skp1p is important for the release of Rav1p from endosomal membranes where it functions in V-ATPase assembly. Thus, although part of the RAVE complex, Skp1p does not appear to be involved in V-ATPase assembly but instead in the cycling of the complex off membranes. This work also provides a generalizable approach to defining the roles of interactions of Skp1p with individual F-box proteins through the isolation of special alleles of SKP1.     

Human exonuclease I is required for 5' and 3' mismatch repair.

We have partially purified a human activity that restores mismatch-dependent, bi-directional excision to a human nuclear extract fraction depleted for one or more mismatch repair excision activities. Human EXOI co-purifies with the excision activity, and the purified activity can be replaced by near homogeneous recombinant hEXOI. Despite the reported 5' to 3' hydrolytic polarity of this activity, hEXOI participates in mismatch-provoked excision directed by a strand break located either 5' or 3' to the mispair. When the strand break that directs repair is located 3' to the mispair, hEXOI- and mismatch-dependent gap formation in excision-depleted extracts requires both hMutSalpha and hMutLalpha. However, excision directed by a 5' strand break requires hMutSalpha but can occur in absence of hMutLalpha. In systems comprised of pure components, the 5' to 3' hydrolytic activity of hEXOI is activated by hMutSalpha in a mismatch-dependent manner. These observations indicate a hydrolytic function for hEXOI in 5'-heteroduplex correction. The involvement of hEXOI in 3'-heteroduplex repair suggests that it has a regulatory/structural role in assembly of the 3'-excision complex or that the protein possesses a cryptic 3' to 5' hydrolytic activity.     

p53 is required for chloroquine-induced atheroprotection but not insulin sensitization

An intact genotoxic stress response appears to be atheroprotective and insulin sensitizing. ATM, mutated in ataxia telangiectasia, is critical for the genotoxic stress response, and its deficiency is associated with accelerated atherosclerosis and insulin resistance in humans and mice. The antimalarial drug chloroquine activates ATM signaling and improves metabolic phenotypes in mice. p53 is a major effector of ATM signaling, but it is unknown if p53 is required for the beneficial effects of chloroquine. We tested the hypothesis that the cardiometabolic effects of chloroquine are p53-dependent. ApoE-null mice with or without p53 were treated with low-dose chloroquine or saline in the setting of a Western diet. After 8 weeks, there was no p53-dependent or chloroquine-specific effect on serum lipids or body weight. Chloroquine reduced plaque burden in mice wild-type for p53, but it did not decrease lesion extent in p53-null mice. However, chloroquine improved glucose tolerance, enhanced insulin sensitivity, and increased hepatic Akt signaling regardless of the p53 genotype. These results indicate that atheroprotection induced by chloroquine is p53-dependent but the insulin-sensitizing effects of this agent are not. Discrete components of the genotoxic stress response might be targeted to treat lipid-driven disorders, such as diabetes and atherosclerosis.     

ATR is not required for p53 activation but synergizes with p53 in the replication checkpoint.

ATR (ataxia telangiectasia and Rad-3-related) is a protein kinase required for survival after DNA damage. A critical role for ATR has been hypothesized to be the regulation of p53 and other cell cycle checkpoints. ATR has been shown to phosphorylate p53 at Ser(15), and this damage-induced phosphorylation is diminished by expression of a catalytically inactive (ATR-kd) mutant. p53 function could not be examined directly in prior studies of ATR, however, because p53 was mutant or because cells expressed the SV40 large T antigen that blocks p53 function. To test the interactions of ATR and p53 directly we generated human U2OS cell lines inducible for either wild-type or kinase-dead ATR that also have an intact p53 pathway. Indeed, ATR-kd expression sensitized these cells to DNA damage and caused a transient decrease in damage-induced serine 15 phosphorylation of p53. However, we found that the effects of ATR-kd expression do not result in blocking the response of p53 to DNA damage. Specifically, prior ATR-kd expression had no effect on DNA damage-induced p53 protein up-regulation, p53-DNA binding, p21 mRNA up-regulation, or G(1) arrest. Instead of promoting survival via p53 regulation, we found that ATR protects cells by delaying the generation of mitotic phosphoproteins and inhibiting premature chromatin condensation after DNA damage or hydroxyurea. Although p53 inhibition (by E6 or MDM2 expression) had little effect on premature chromatin condensation, when combined with ATR-kd expression there was a marked loss of the replication checkpoint. We conclude that ATR and p53 can function independently but that loss of both leads to synergistic disruption of the replication checkpoint.     

Substrate recognition by a eukaryotic RNase III: the double-stranded RNA-binding domain of Rnt1p selectively binds RNA containing a 5'-AGNN-3' tetraloop

Rnt1p is an RNase III homolog from budding yeast, required for processing snRNAs, snoRNAs, and rRNA. Numerous Rnt1p RNA substrates share potential to form a duplex structure with a terminal four-base loop with the sequence AGNN. Using a synthetic RNA modeled after the 25S rRNA 3' ETS cleavage site we find that the AGNN loop is an important determinant of substrate selectivity. When this loop sequence is altered, the rate of Rnt1p cleavage is reduced. The reduction in cleavage rate can be attributed to reduced binding of the mutant substrate as measured by a gel-shift assay. Deletion of the nonconserved N-terminal domain of Rnt1p does not affect cleavage site choice or the ability of the enzyme to distinguish substrates that contain the AGNN loop, indicating that this region is not required for selective cleavage. Strikingly, a recombinant fragment of Rnt1p containing little more than the dsRBD is able to discriminate between wild-type and mutant loop sequences in a binding assay. We propose that a major determinant of AGNN loop recognition by Rnt1p is present in its dsRBD.     

p53 is required for nuclear but not mitochondrial DNA damage-induced degeneration

While the consequences of nuclear DNA damage have been well studied, the exact consequences of acute and selective mitochondrial DNA (mtDNA) damage are less understood. DNA damaging chemotherapeutic drugs are known to activate p53-dependent apoptosis in response to sustained nuclear DNA damage. While it is recognized that whole-cell exposure to these drugs also damages mtDNA, the specific contribution of mtDNA damage to cellular degeneration is less clear. To examine this, we induced selective mtDNA damage in neuronal axons using microfluidic chambers that allow for the spatial and fluidic isolation of neuronal cell bodies (containing nucleus and mitochondria) from the axons (containing mitochondria). Exposure of the DNA damaging drug cisplatin selectively to only the axons induced mtDNA damage in axonal mitochondria, without nuclear damage. We found that this resulted in the selective degeneration of only the targeted axons that were exposed to DNA damage, where ROS was induced but mitochondria were not permeabilized. mtDNA damage-induced axon degeneration was not mediated by any of the three known axon degeneration pathways: apoptosis, axon pruning, and Wallerian degeneration, as Bax-deficiency, or Casp3-deficiency, or Sarm1-deficiency failed to protect the degenerating axons. Strikingly, p53, which is essential for degeneration after nuclear DNA damage, was also not required for degeneration induced with mtDNA damage. This was most evident when the p53-deficient neurons were globally exposed to cisplatin. While the cell bodies of p53-deficient neurons were protected from degeneration in this context, the axons farthest from the cell bodies still underwent degeneration. These results highlight how whole cell exposure to DNA damage activates two pathways of degeneration; a faster, p53-dependent apoptotic degeneration that is triggered in the cell bodies with nuclear DNA damage, and a slower, p53-independent degeneration that is induced with mtDNA damage.Subject terms: Cell biology, Neuroscience     

The Mre11 nuclease is not required for 5' to 3' resection at multiple HO-induced double-strand breaks

Current hypotheses suggest the Mre11 nuclease activity could be directly involved in double-strand break (DSB) resection in the presence of a large number of DSBs or limited to processing abnormal DNA ends. To distinguish between these possibilities, we used two methods to create large numbers of DSBs in Saccharomyces cerevisiae chromosomes, without introducing other substrates for the Mre11 nuclease. Multiple DSBs were created either by expressing the HO endonuclease in strains containing several HO cut sites embedded within randomly dispersed Ty1 elements or by phleomycin treatment. Analysis of resection by single-strand DNA formation in these systems showed no difference between strains containing MRE11 or the mre11-D56N nuclease defective allele, suggesting that the Mre11 nuclease is not involved in the extensive 5' to 3' resection of DSBs. We postulate that the ionizing radiation (IR) sensitivity of mre11 nuclease-defective mutants results from the accumulation of IR-induced DNA damage that is normally processed by the Mre11 nuclease. We also report that the processivity of 5' to 3' DSB resection and the yield of repaired products are affected by the number of DSBs in a dose-dependent manner. Finally, we show that the exonuclease Exo1 is involved in the processivity of 5' to 3' resection of an HO-induced DSB at the MAT locus.     

Poliovirus CRE-dependent VPg uridylylation is required for positive-strand RNA synthesis but not for negative-strand RNA synthesis

The cis-acting replication element (CRE) is a 61-nucleotide stem-loop RNA structure found within the coding sequence of poliovirus protein 2C. Although the CRE is required for viral RNA replication, its precise role(s) in negative- and positive-strand RNA synthesis has not been defined. Adenosine in the loop of the CRE RNA structure functions as the template for the uridylylation of the viral protein VPg. VPgpUpU(OH), the predominant product of CRE-dependent VPg uridylylation, is a putative primer for the poliovirus RNA-dependent RNA polymerase. By examining the sequential synthesis of negative- and positive-strand RNAs within preinitiation RNA replication complexes, we found that mutations that disrupt the structure of the CRE prevent VPg uridylylation and positive-strand RNA synthesis. The CRE mutations that inhibited the synthesis of VPgpUpU(OH), however, did not inhibit negative-strand RNA synthesis. A Y3F mutation in VPg inhibited both VPgpUpU(OH) synthesis and negative-strand RNA synthesis, confirming the critical role of the tyrosine hydroxyl of VPg in VPg uridylylation and negative-strand RNA synthesis. trans-replication experiments demonstrated that the CRE and VPgpUpU(OH) were not required in cis or in trans for poliovirus negative-strand RNA synthesis. Because these results are inconsistent with existing models of poliovirus RNA replication, we propose a new four-step model that explains the roles of VPg, the CRE, and VPgpUpU(OH) in the asymmetric replication of poliovirus RNA.