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Functional RNAs; RNA catalysts, miRNA, sRNA, RNAi...

10/26/09. Kevin Klein, PhD. Slide 2. RNAs have many functions. rRNA (ribosomal RNA)tRNA (transfer RNA)mRNA (Messenger RNA)snRNA (including snoRNA) (Small nuclear RNA- splicing)Other ribozymesTelomerase RNA (telomerase maintenance)siRNA (small interfering RNA RNA silencing)shRNA (short hairp

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Functional RNAs; RNA catalysts, miRNA, sRNA, RNAi...

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    1. Functional RNAs; RNA catalysts, miRNA, sRNA, RNAi... Kevin Klein, PhD. Pathobiology 551 Monday 10/26/2009

    2. 10/26/09 Kevin Klein, PhD Slide 2 RNAs have many functions rRNA (ribosomal RNA) tRNA (transfer RNA) mRNA (Messenger RNA) snRNA (including snoRNA) (Small nuclear RNA- splicing) Other ribozymes Telomerase RNA (telomerase maintenance) siRNA (small interfering RNA – RNA silencing) shRNA (short hairpin – RNA silencing) Pri-miRNA; miRNA ( (primary) micro RNA - regulation) sRNA (small RNA – bacteria) gRNA (guide RNA – RNA editing) Genomic RNA (viruses)

    3. 10/26/09 Kevin Klein, PhD Slide 3 “Regulatory” RNAs in prokaryotes Can Regulate at the level of... mRNA production (transcriptional attenuation) mRNA expression (translational initiation) mRNA stability (mRNA degradation)

    4. 10/26/09 Kevin Klein, PhD Slide 4 “Ribozymes” Cleave RNA Can carry out other reactions. Example??? Can regulate mRNA expression Function in both prokaryotes and eukaryotes. Can be associated with large ribonucleoprotein complexes (RNP) Can be used experimentally for knockdowns Difficult to get a good knockdown using ribozymes.

    5. 10/26/09 Kevin Klein, PhD Slide 5 Ribozymes: Ribonucleic Acid Enzymes

    6. 10/26/09 Kevin Klein, PhD Slide 6 Hammerhead ribozyme Smallest known ribozyme (40 bp) Associated with plant viroid genome replication

    7. 10/26/09 Kevin Klein, PhD Slide 7

    8. 10/26/09 Kevin Klein, PhD Slide 8

    9. 10/26/09 Kevin Klein, PhD Slide 9 Riboswitches RNA species that can bind metabolites/ions Binding of substrates regulates mRNA expression Transcriptional termination Translation repression/initiation mRNA stability Regulate ~2% B. subtilis genes Also present in Eukaryotes.

    10. 10/26/09 Kevin Klein, PhD Slide 10

    11. 10/26/09 Kevin Klein, PhD Slide 11 Example of a riboswitch (using a ribozyme) glmS – Paper For Discussion

    12. 10/26/09 Kevin Klein, PhD Slide 12 Small RNAs (sRNA or saRNA) Genomically encoded (intergenic regions) Frequently are complimentary to mRNA Usually imperfect complementary 80 – 120 (+) nt in length Highly conserved between species Can have multiple unlinked targets Usually interact with the 5’ UTR Frequently require Hfq for function

    13. 10/26/09 Kevin Klein, PhD Slide 13

    14. 10/26/09 Kevin Klein, PhD Slide 14

    15. 10/26/09 Kevin Klein, PhD Slide 15

    16. 10/26/09 Kevin Klein, PhD Slide 16 DsrA - H-NS represses many stress induced genes - rpoS (sigma factor) promotes stationary phase

    17. 10/26/09 Kevin Klein, PhD Slide 17 Small RNAs in Eukaryotes Micro RNAs (miRNA) Similar to sRNAs in prokaryotes Small interfering RNA (siRNA) Induces mRNA degradation

    18. 10/26/09 Kevin Klein, PhD Slide 18

    19. 10/26/09 Kevin Klein, PhD Slide 19 miRNA structures

    20. 10/26/09 Kevin Klein, PhD Slide 20 miRNA Processing In nucleus, Drosha-DCGR8 cleaves stem-loop pri-miRNA liberating a ~70 nt hairpin (pre-miRNA) Pre-miRNA is exported to the cytoplasm Exportin-5 and RAN-GTP dependent In the cytoplasm, Dicer cleaves liberating a ~22 nucleotide RNA duplex with 2 bp 3’overhangs.

    21. 10/26/09 Kevin Klein, PhD Slide 21 Drosha and Dicer Members of the RNase III family dsRNA-specific Cleave dsRNA leaving 2 bp 3’ overhangs

    22. 10/26/09 Kevin Klein, PhD Slide 22 miRNA processing

    23. 10/26/09 Kevin Klein, PhD Slide 23 miRNA function Mechanism of action is unknown Interacts with the 3’UTR of the target mRNA Inhibits translation from a mRNA without inducing mRNA degradation Can form a ribonucleoprotein complex (miRNP) Can be complexed with ribosomes and target mRNA Also important for silencing retrotransposons and endogenous retroviruses Important for development and differentiation miRNA:mRNA (miRNP) targeted to p-bodies

    24. 10/26/09 Kevin Klein, PhD Slide 24

    25. 10/26/09 Kevin Klein, PhD Slide 25 miRNA and Viruses miRNA are encoded by viruses Mostly nuclear replicating DNA viruses (herpesviruses) Can target either viral or host mRNAs Frequently are associated in latency Still controversial whether RNA viruses encode miRNA Why wouldn’t they? Host miRNA can target viral mRNA or genomic RNA Can be either pro-virus or antivirus MiR-122 facilitates HCV replication HIV latency in resting PBMCs (miR-28, miR-125b, miR-150, miR-223 and miR-382 ) Mir-32 can inhibit HFV replication

    26. 10/26/09 Kevin Klein, PhD Slide 26 Virus encoded miRNA targeting …

    27. 10/26/09 Kevin Klein, PhD Slide 27 siRNA... Originally characterized in plants “Post transcriptional gene silencing” Plays a large role in plant “immune system” Subsequently found in a wide variety of eukaryotes Fire and Mello successfully silenced genes in C. elegans 1998 (won Nobel in 2006) Triggered by dsRNA

    28. 10/26/09 Kevin Klein, PhD Slide 28 siRNA Similar to miRNA. BUT... Typically induces mRNA degradation Requires exact base pairing for maximum effect Dicer processes double stranded RNAs RISC (or other complexes) carry out the effector function RISC cleaves RNA that is complementary to dsRNA that activates pathway This url contains an animated video clip of RNAi

    29. 10/26/09 Kevin Klein, PhD Slide 29 GFP silencing in transgenic C. elegans

    30. 10/26/09 Kevin Klein, PhD Slide 30 RISC effector complex Contains a member of the argonaute family Between 130 kDa and 500 kDa Other components are being characterized Cleaves RNA complementary to the siRNA, in the middle of the sequence Assembling the RISC complex requires ATP, while RNA cleavage does not.

    31. 10/26/09 Kevin Klein, PhD Slide 31 Uses of siRNA Gene knockdowns Look at function/phenotype of a gene Therapeutic techniques Anti viral Anti cancer Anti neurological diseases Others

    32. 10/26/09 Kevin Klein, PhD Slide 32 siRNA – designing the assay dsRNAs need to be <30 bp in length Why? Well-designed siRNAs can result in >90% reduction in target RNA 21nt dsRNAs most effective Sequence-specificity important Single bp-mismatches reduce silencing capability Many will make 3-4 siRNAs – test all and go w/ best Deliver by injection or transfection Vectors becoming more popular

    33. 10/26/09 Kevin Klein, PhD Slide 33 Caveats of siRNA Not found in apicomplexans Exceptions to the rule Scattered literature in P. falciparum but not reproducible T. brucei – yes T. cruzi and Leishmania -- no

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