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Primary structure of 53BP1

Primary structure of 53BP1. 1. BRCT. BRCT. 1957. 1220. RG. TUDOR. TUDOR. NLS. 1703. 53BP1 and RNAs. RNA-dependent recruitment to foci following DNA damage Strong RNA binding activity Interacts with 7SL RNA the ancestor of SINE B1 (mouse) and Alu (human) sequences.

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Primary structure of 53BP1

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  1. Primary structure of 53BP1 1 BRCT BRCT 1957 1220 RG TUDOR TUDOR NLS 1703

  2. 53BP1 and RNAs • RNA-dependent recruitment to foci following DNA damage • Strong RNA binding activity • Interacts with 7SL RNA the ancestor of SINE B1 (mouse) and Alu (human) sequences

  3. Generation of 53BP1 knock-down cellsusing ShRNAs Quantitative RT-PCR Western blot 53BP1 actin mES ShA ShB

  4. IP pAb53BP1+(E14T+Sh53BP1) Input E14T+Sh53BP1 (5%) Input E14T (5%) Control IgG+E14T pAb53BP1+E14T 53BP1 - Specificity of the anti-53BP1 antibody used in IP experiences

  5. Purification of small RNAs associated with 53BP1 (1) ES cells Formalin crosslinked Supernatant pellet Extraction with 350mM NaCl Supernatant pellet IP 53BP1 Reverse crosslink +RNA extraction

  6. Purification of small RNAs associated with 53BP1 (2) RNA Immunoprecipitation ES Sh53BP1 (ShA) ES • Cloning • Sequencing • -Bioinformatics analysis

  7. Bioinformatics analysis Ex: Chromosome 1

  8. Small nucleolar RNA (snoRNA) are involved in rRNA maturation Pseudouridylation of rRNA Methylation of rRNA

  9. D’box C’box C box D box 5’ 3’ 1-17 are missing in the library snord79

  10. snoRNAs and Small Derived RNAs (sdRNAs)(Taft et al., 2009) • More than 60% of SnoRNAs give rise to sdRNAs • sdRNAs are highly conserved • C/D box sdRNAs are predominantly 17-19nt and 30nt, while H/ACA box sdRNAs are 22nt • sdRNAs are only midly down-regulated in dicer1- and dgcr8-deficient ES cells. = Is 53BP1 involved in the generation of sdRNAs ??

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