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The Analysis of Pendolino (peo) Mutants Reveals Differences in the Fusigenic Potential among Drosophila Telomeres.

Cenci G, Ciapponi L, Marzullo M, Raffa GD, Morciano P, Raimondo D, Burla R, Saggio I, Gatti M - PLoS Genet. (2015)

Bottom Line: The Peo protein directly interacts with the terminin components, but peo mutations do not affect telomeric localization of HOAP, Moi, Ver and HP1a, suggesting that the peo-dependent telomere fusion phenotype is not due to loss of terminin from chromosome ends. peo mutants are also defective in DNA replication and PCNA recruitment.However, our results suggest that general defects in DNA replication are unable to induce TFs in Drosophila cells.We thus hypothesize that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in heterochromatin-associated telomeres.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biologia e Biotecnologie, Sapienza-Università di Roma, Roma, Italy; Istituto Pasteur Fondazione Cenci Bolognetti, Sapienza-Università di Roma, Roma, Italy.

ABSTRACT
Drosophila telomeres are sequence-independent structures that are maintained by transposition to chromosome ends of three specialized retroelements (HeT-A, TART and TAHRE; collectively designated as HTT) rather than telomerase activity. Fly telomeres are protected by the terminin complex (HOAP-HipHop-Moi-Ver) that localizes and functions exclusively at telomeres and by non-terminin proteins that do not serve telomere-specific functions. Although all Drosophila telomeres terminate with HTT arrays and are capped by terminin, they differ in the type of subtelomeric chromatin; the Y, XR, and 4L HTT are juxtaposed to constitutive heterochromatin, while the XL, 2L, 2R, 3L and 3R HTT are linked to the TAS repetitive sequences; the 4R HTT is associated with a chromatin that has features common to both euchromatin and heterochromatin. Here we show that mutations in pendolino (peo) cause telomeric fusions (TFs). The analysis of several peo mutant combinations showed that these TFs preferentially involve the Y, XR and 4th chromosome telomeres, a TF pattern never observed in the other 10 telomere-capping mutants so far characterized. peo encodes a non-terminin protein homologous to the E2 variant ubiquitin-conjugating enzymes. The Peo protein directly interacts with the terminin components, but peo mutations do not affect telomeric localization of HOAP, Moi, Ver and HP1a, suggesting that the peo-dependent telomere fusion phenotype is not due to loss of terminin from chromosome ends. peo mutants are also defective in DNA replication and PCNA recruitment. However, our results suggest that general defects in DNA replication are unable to induce TFs in Drosophila cells. We thus hypothesize that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in heterochromatin-associated telomeres. Alternatively, it is possible that Peo plays a dual function being independently required for DNA replication and telomere capping.

No MeSH data available.


Related in: MedlinePlus

Mapping the Peo regions that interact with terminin.(A) A tridimensional molecular model for Peo. The arrow pointing to “N-ter” indicates the N-terminus of the protein; the arrow pointing to “C-ter” indicates the starting site of the disordered C-terminal region of Peo (not depicted); the variant Asp residue and His-Pro-His motif are represented as sticks and indicated by red and purple arrows, respectively (see Materials and Methods and S2 Fig for construction of the Peo 3D model). (B) Schematic organization of the Peo protein and Peo truncations used for GST pulldown. (C-E) GST-pulldown from S2 cells extracts expressing HOAP-FLAG (C), Ver-FLAG (D) or Moi-HA (E). HOAP-FLAG, Ver-FLAG and Moi-HA were detected with anti-FLAG and anti-HA antibodies. The C-terminal disordered region (included in the Peo 3 fragment) does not interact with any of the terminin components. HOAP specifically interacts with N-terminal region of Peo; in contrast, Moi and Ver interact with both the N terminal and the UEV-containing central regions of the protein.
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pgen.1005260.g006: Mapping the Peo regions that interact with terminin.(A) A tridimensional molecular model for Peo. The arrow pointing to “N-ter” indicates the N-terminus of the protein; the arrow pointing to “C-ter” indicates the starting site of the disordered C-terminal region of Peo (not depicted); the variant Asp residue and His-Pro-His motif are represented as sticks and indicated by red and purple arrows, respectively (see Materials and Methods and S2 Fig for construction of the Peo 3D model). (B) Schematic organization of the Peo protein and Peo truncations used for GST pulldown. (C-E) GST-pulldown from S2 cells extracts expressing HOAP-FLAG (C), Ver-FLAG (D) or Moi-HA (E). HOAP-FLAG, Ver-FLAG and Moi-HA were detected with anti-FLAG and anti-HA antibodies. The C-terminal disordered region (included in the Peo 3 fragment) does not interact with any of the terminin components. HOAP specifically interacts with N-terminal region of Peo; in contrast, Moi and Ver interact with both the N terminal and the UEV-containing central regions of the protein.

Mentions: peo encodes an E2 variant (UEV) enzyme; the UEV proteins are similar to the E2 ubiquitin conjugating enzymes (UBCs) but lack the catalytic cysteine residue that mediates the interaction between ubiquitin and E2 [40]. We elaborated a three-dimensional model of Peo exploiting a series of bionformatic analyses (Fig 6A; see also Material and Methods and S2 Fig). We confirmed that Peo lacks the catalytic cysteine of E2 enzymes. In addition, 8 residues before the catalytic cysteine site, Peo exhibits an HPH tripeptide (S2 Fig) instead of HPN, which is a canonical signature of the E2 superfamily [41]. Peo contains a UEV domain of ~150 amino acids resembling the canonical E2 fold in its hydrophobic core and active site region. This domain consists of three helices packed against a four-stranded antiparallel β-sheet. Next to the UEV domain, Peo contains two C-terminal helices that are present in all E2 proteins but missing in other E2 variant enzymes such as Tsg101 and Mms2. Finally, prediction of potentially disordered regions revealed that Peo also contains a long (50 aa) disordered region at the C-terminus (Figs 6A and S2; see also Materials and Methods).


The Analysis of Pendolino (peo) Mutants Reveals Differences in the Fusigenic Potential among Drosophila Telomeres.

Cenci G, Ciapponi L, Marzullo M, Raffa GD, Morciano P, Raimondo D, Burla R, Saggio I, Gatti M - PLoS Genet. (2015)

Mapping the Peo regions that interact with terminin.(A) A tridimensional molecular model for Peo. The arrow pointing to “N-ter” indicates the N-terminus of the protein; the arrow pointing to “C-ter” indicates the starting site of the disordered C-terminal region of Peo (not depicted); the variant Asp residue and His-Pro-His motif are represented as sticks and indicated by red and purple arrows, respectively (see Materials and Methods and S2 Fig for construction of the Peo 3D model). (B) Schematic organization of the Peo protein and Peo truncations used for GST pulldown. (C-E) GST-pulldown from S2 cells extracts expressing HOAP-FLAG (C), Ver-FLAG (D) or Moi-HA (E). HOAP-FLAG, Ver-FLAG and Moi-HA were detected with anti-FLAG and anti-HA antibodies. The C-terminal disordered region (included in the Peo 3 fragment) does not interact with any of the terminin components. HOAP specifically interacts with N-terminal region of Peo; in contrast, Moi and Ver interact with both the N terminal and the UEV-containing central regions of the protein.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4481407&req=5

pgen.1005260.g006: Mapping the Peo regions that interact with terminin.(A) A tridimensional molecular model for Peo. The arrow pointing to “N-ter” indicates the N-terminus of the protein; the arrow pointing to “C-ter” indicates the starting site of the disordered C-terminal region of Peo (not depicted); the variant Asp residue and His-Pro-His motif are represented as sticks and indicated by red and purple arrows, respectively (see Materials and Methods and S2 Fig for construction of the Peo 3D model). (B) Schematic organization of the Peo protein and Peo truncations used for GST pulldown. (C-E) GST-pulldown from S2 cells extracts expressing HOAP-FLAG (C), Ver-FLAG (D) or Moi-HA (E). HOAP-FLAG, Ver-FLAG and Moi-HA were detected with anti-FLAG and anti-HA antibodies. The C-terminal disordered region (included in the Peo 3 fragment) does not interact with any of the terminin components. HOAP specifically interacts with N-terminal region of Peo; in contrast, Moi and Ver interact with both the N terminal and the UEV-containing central regions of the protein.
Mentions: peo encodes an E2 variant (UEV) enzyme; the UEV proteins are similar to the E2 ubiquitin conjugating enzymes (UBCs) but lack the catalytic cysteine residue that mediates the interaction between ubiquitin and E2 [40]. We elaborated a three-dimensional model of Peo exploiting a series of bionformatic analyses (Fig 6A; see also Material and Methods and S2 Fig). We confirmed that Peo lacks the catalytic cysteine of E2 enzymes. In addition, 8 residues before the catalytic cysteine site, Peo exhibits an HPH tripeptide (S2 Fig) instead of HPN, which is a canonical signature of the E2 superfamily [41]. Peo contains a UEV domain of ~150 amino acids resembling the canonical E2 fold in its hydrophobic core and active site region. This domain consists of three helices packed against a four-stranded antiparallel β-sheet. Next to the UEV domain, Peo contains two C-terminal helices that are present in all E2 proteins but missing in other E2 variant enzymes such as Tsg101 and Mms2. Finally, prediction of potentially disordered regions revealed that Peo also contains a long (50 aa) disordered region at the C-terminus (Figs 6A and S2; see also Materials and Methods).

Bottom Line: The Peo protein directly interacts with the terminin components, but peo mutations do not affect telomeric localization of HOAP, Moi, Ver and HP1a, suggesting that the peo-dependent telomere fusion phenotype is not due to loss of terminin from chromosome ends. peo mutants are also defective in DNA replication and PCNA recruitment.However, our results suggest that general defects in DNA replication are unable to induce TFs in Drosophila cells.We thus hypothesize that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in heterochromatin-associated telomeres.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biologia e Biotecnologie, Sapienza-Università di Roma, Roma, Italy; Istituto Pasteur Fondazione Cenci Bolognetti, Sapienza-Università di Roma, Roma, Italy.

ABSTRACT
Drosophila telomeres are sequence-independent structures that are maintained by transposition to chromosome ends of three specialized retroelements (HeT-A, TART and TAHRE; collectively designated as HTT) rather than telomerase activity. Fly telomeres are protected by the terminin complex (HOAP-HipHop-Moi-Ver) that localizes and functions exclusively at telomeres and by non-terminin proteins that do not serve telomere-specific functions. Although all Drosophila telomeres terminate with HTT arrays and are capped by terminin, they differ in the type of subtelomeric chromatin; the Y, XR, and 4L HTT are juxtaposed to constitutive heterochromatin, while the XL, 2L, 2R, 3L and 3R HTT are linked to the TAS repetitive sequences; the 4R HTT is associated with a chromatin that has features common to both euchromatin and heterochromatin. Here we show that mutations in pendolino (peo) cause telomeric fusions (TFs). The analysis of several peo mutant combinations showed that these TFs preferentially involve the Y, XR and 4th chromosome telomeres, a TF pattern never observed in the other 10 telomere-capping mutants so far characterized. peo encodes a non-terminin protein homologous to the E2 variant ubiquitin-conjugating enzymes. The Peo protein directly interacts with the terminin components, but peo mutations do not affect telomeric localization of HOAP, Moi, Ver and HP1a, suggesting that the peo-dependent telomere fusion phenotype is not due to loss of terminin from chromosome ends. peo mutants are also defective in DNA replication and PCNA recruitment. However, our results suggest that general defects in DNA replication are unable to induce TFs in Drosophila cells. We thus hypothesize that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in heterochromatin-associated telomeres. Alternatively, it is possible that Peo plays a dual function being independently required for DNA replication and telomere capping.

No MeSH data available.


Related in: MedlinePlus