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CRISPR-associated Proteins - PowerPoint PPT Presentation

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CRISPR-associated Proteins

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  1. CRISPR-associated Proteins Sarah Pyfrom sapyfrom@davidson.edu

  2. Research Questions • What Cas-proteins does our species share with the 10 other species we chose to study? • If so, how do they compare? • How do Cas-proteins function in relation to CRISPR units? [Edit]: • Why did JGI change its annotation?

  3. Cas Proteins • Proteins that are almost always associated with (near) CRISPR sequences • Originally four major families • Now, at least 45 families total

  4. JGI annotation “Old” Cas-Proteins “New” Cas-Proteins Cas1 Cas2 Cas3 Cas4 TM1800 TM1801 Cas1 Cas2 Cas4 Cas5 Cas6 Csh1 Csh2

  5. Changes: • TM1800= Cas5 • TM1801=Csh2 • Hypothetical protein = Csh1 • Part of hypothetical protein = Cas6 • Cas3 = hypothetical protein • Cas4: • MTDSSGDPVDRFLAAARDESAELPFRLTGVMFQYYVVCERELWFLSRDVEIDRDTPAIVRGSDVDDSAYADKRRDVRVDGIIAIDVLDSGEILEVKPSSSMTEPARLQLLFYLWYLDRVTGVEKTGVLAHPAEKRRETVELTPETSAEVESAIEGIRAVVTAESPPPAEEKPVCDSCAYHDFCWSC (red = original Cas4)

  6. Map of CRISPR region TM1800 TM1801 Transposases Cas3 Hypothetical proteins CRISPR Cas1 Unidentified Csh1 Cas2 Cas5 Cas4 Cas6 Csh2

  7. Cas1(from Sulfolobus solfataricus) • high-affinity nucleic acid binding protein • binds DNA, RNA and DNA–RNA hybrid • sequence non-specific in a multi-site binding mode • promotes the hybridization of complementary nucleic acid strands. • From: SSO1450 – A CAS1 protein from Sulfolobus solfataricus P2 with high affinity for RNA and DNA

  8. Cas2 function unknown Cas3 Cas4 Usually similar to helicases Unwinds double-stranded DNA Thought to be involved in DNA metabolism and repair Often resemble Rec-B exonucleases Break down nucleic acid strains Thought to be involved in DNA metabolism From Genbank

  9. Cas5 Cas6 Often found with Cas1, and Cas6. Share and N-terminal region of about 43 amino acids in length Are usually 210-265 amino acids long Characterized by GhGxxxxxGhG motif, where h indicates a hydrophobic residue, at the C-terminus From: EMBL IPR013422 profile page (: http://www.ebi.ac.uk/interpro/IEntry?ac=IPR013422) From: Sanger PF09559 Profile page ( http://pfam.sanger.ac.uk/family/PF09559)

  10. Csh1 and Csh2? • Protein families determined for ease of alignment • Often large differences between species • Alignment easier if protein “soup” is divided into more readily-compared subgroups.

  11. CRISPRs thought to create stable secondary RNA structures • Spacers remain associated with their DR neighbors. • Provide a way for Cas-Proteins to recognize the spacers and facilitate immune response. From: Evolutionary conservation of sequence and secondary structures in CRISPR repeats

  12. Cas-Proteins and Immunity • Thought to act like Slicer and Dicer (eukaryotic counterpart) • Create siRNA that will inhibit/break down invading RNA • Not known if Cas-proteins are involved in integrating pathogenic DNA into spacers Video of eukaryotic siRNA process: http://www.youtube.com/watch?v=D-77BvIOLd0

  13. Alignments of Cas • Compared Cas1, Cas2, Cas3 etc. proteins across all 10 species…

  14. Comparison with other species:(based on “old” proteins)

  15. Phylogenetic tree comparing amino acid sequences for all CAS-proteins 2 2 Halomicrobium mukohataei 2 2 Haloarcula sinaiiensis 1800 1 Haloarcula californiae 4 Haloferax dentrificans 3 1 Haloferax mediteranei 1801 1801 Haloferax sulfurifontis 1801 1801 Haloferax mucosum 1801 Halorhabdus utahensis 3 1 1 1800 2 3 3 2 1800 1 1800 1800 1 3 1801 4 4 4 4 4 1 4 1800 3 3 1800 1801

  16. Cas 1 and Cas2 did not change

  17. Cas 4 • JGI revision shortened this protein • Would expect low sequence similarity near end of protein

  18. TM1801 (Csh2) • Revision by JGI simply renamed this protein • Would expect sequence similarity

  19. Map of CRISPR region TM1800 TM1801 Transposases Cas3 Hypothetical proteins CRISPR Cas1 Unidentified Csh1 Cas2 Cas5 Cas4 Cas6 Csh2

  20. In conclusion: We don’t know much…. …but we do know everything that everybody else knows.

  21. Questions?

  22. References • Kunin, V., Sorek, R., Hugenholtz, P. (2007) Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biology.http://genomebiology.com/2007/8/4/R61. Accessed 24 Nov, 2009. • Haft, D.H., Selengut, J. Mongodin, EF., Nelson, K.E. (2005). A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput Biol. http://www.ncbi.nlm.nih.gov/pubmed/16292354. Accessed 24 Nov 2009.