New microribbon production

Transcription

1 New microribbon production In vitro transformation Excystation brief exposure to acidic ph (~2) flagellar activity within 5-10 min after return to neutral ph breakdown of cyst wall (proteases) trophozoite emerges from cyst cytokinesis within 30 min Encystation exposure to ph 7, no bile exposure to ph 7.8, high bile cyst wall secretion (appearance of vesicles) loss of disk and flagella nuclear division

2 Golgi reorganization - early Trophozoite - No visible golgi via EM Encystation Early stages Major cytoskeletal reorganization Stacked tubular structures Peripheral vacuoles Rapid synthesis of CWP1 and CWP2 Breaking of ventral disk and flagella - chunks Golgi reorganization - late Secretion Vesicular transport to membrane ESV - encystation stage vesicles CWP1 and CWP2 NO CHITIN Major sugar component N-acetyl-galactosamine Biosynthesis is upregulated during encystation process Learning Objectives Define RNA interference Define basic terminology Describe molecular mechanism Define VSP and relevance Describe role of RNAi in antigenic variation

3 A Nobel Way to Regulate Gene Expression Current number of reviews (2007) (2008) (2009 Feb) (2009 Oct) ~8684 DNA replication reviews 8 years following initial discovery in C. elegans: the Nobel prize was awarded to Andy Fire and Craig Mello!! Andy Fire Stanford for their discovery of RNA interference: gene silencing by double-stranded RNA" Craig Mello UMass Med 2006 Nobel Prize in Physiology or Medicine Experimental Expectations A Perplexing Result Loss of Pigment Result: Hypopigmentation

4 What is RNA interference? Way to silence gene expression - post-transcriptional dsrna does not produce global down regulation, but is sequence specific - leads to the specific degradation of a corresponding mrna Gene silencing - no need to delete the gene Conserved pathway thought to be involved in retrotransposon and viral defense Now, gene regulatory mechanisms involving small RNAs Discovery of RNAi First discovered in C. elegans by Andy Fire et al (1998). Coined the term RNA interference Same year, initial observations of similar phenomenon in T. brucei by Elisabetta Ullu et al. Both sense and antisense RNAs were sufficient for silencing.how??? Silencing can persist and is potent Hugely HOT topic New information every month Increasingly detailed knowledge Large body of literature Stick to the basics Terminology - RNA Players dsrna: double stranded RNA longer than 30 nt sirna: short interfering RNA, nt Mostly exogenous origin (synthetic RNAs) dsrna precursors (may be stemloop) Target specific (1 sirna = 1 target) mirna: microrna, nt Encoded by endogenous genes Hairpin precursors Recognize multiple targets (1 mirna = `5 targets)

5 Regulation by small RNAs Exogenous vs. endogenous Share a common pathway initiation End results: Regulation via mrna degradation (sirna) Regulation via translational repression (mirna) execution Key Steps Models for RNAi Processing dsrnas are cleaved into nt fragments = sirna Amplification Recognition sirna (antisense) guides cleavage of specific mrna RNAi Players - simplified Dicer ATP-dependent ribonuclease 2 RNase III domains sirna RISC RNAi silencing complex Contains sirna Directs complex to specific mrna Antisense strand mediated Promotes cleavage of mrna in the middle of sirna

6 Mechanism of RNA interference dsrna Native sirna duplex 5ʼ 5ʼ small interfering RNAs (sirnas) Active sirna duplex (inactive, kDa complex) (a critical step in the activation of RISC) RISC RNA-induced silencing complex (active, 100kDa complex) (endonucleolytic cleavage in the region of homology) Zamore, P. D. Science 296, (2002) RNAi as an experimental tool dsrna (relative long dsrna molecules) Injected into cells - not heritable C. elegans could be fed bacteria containing plasmids that synthesized dsrna DOES NOT work with mammalian cells dsrna (long) induces interferon system leading to nonspecific cytotoxic effect sirna Injected into cells - not heritable - lose effect over cell generations Vectors for cell specific situations Long term steady expression Therapeutic applications Experimental induction of RNAi

7 Giardia RNAi Paper Discussion Papers are loaded with huge amounts of information What should you take away from the paper? Identify a Hypothesis List the Major Finding(s) Learn some Techniques Supplemental - VSG clonality VSP9B10 Green - VSP9B10 antibody Blue - DAPI Demonstration that cell line expresses a single VSP Variant-specific surface protein VSPs are Cysteine-rich % cysteine (disulfide linkages) CXXC motifs usually associated with DNA binding proteins VSP repertoire of possible variants only ~190 expressed Variable N- terminus, Conserved C-terminus. Possibly all VSP genes are transcribed, but ONLY 1 protein is stable. VSP switches every 6-13 generations

8 First Question Question: Which VSPs are transcribed? Methods: Nuclear Run-on vs. Northern blotting Results: Most VSP genes are transcribed Steady state conditions indicate a single stable VSP transcript Low molecular weight RNAs detected Nuclear Run-on vs. Northern Blot Isolate nuclei from cells, allow them to extend in vitro the transcripts already started in vivo in a technique called run-on transcription RNA polymerase that has already initiated transcription will run-on or continue to elongate same RNA chains Effective as initiation of new RNA chains in isolated nuclei does not generally occur. Isolate total RNA from cells Population of molecules includes processed mrna, trna and rrna.. Newly synthesized RNA Steady-state RNA levels Giardia RNAi - Figure 1 Several VSP genes are simultaneously transcribed in Giardia. Nuclear Run-on Northern Nuclear Run-on was used as probe Only one VSP transcript accumulates What happens to the remainder of the VSP transcripts? CG Prucca et al. Nature 456, (2008) doi: /nature07585

9 Dicer is a Conserved PTGS Protein Hypothesized that other VSP transcripts are specifically degraded, a feature of post-transcriptional gene silencing. Next Question Question: Does Giardia express PTGS protein components? Methods: Epitope tagging and detection by immunofluorescence Results: RdRP - subnuclear localization active protein Dicer - cytoplasmic localization Ago - cytoplasmic localization Supplementary - RNAi components * No enzyme control * RdRP activity No requirement for priming (B,C) charactersitic of dsrna mediated reactions RdRP Localization Activity Dicer Localization Argonaute (Ago) Localization The three RNAi components are contitutively expressed in both life cycle stages.

10 Next Question Question: Do Giardia Dicer produce nt products with similar requirements as other systems? Methods: Incubation of Giardia extracts with specific radiolabeled RNA molecules. Results: Small nt products are produced Requirement of ATP demonstrated with an ATP regenerating system. Giardia RNAi - Figure 2 Dicer activity and detection of VSP small RNAs in Giardia. Isolated nuclear extracts active on in vitro synthesized RNAs ATP required for dsrna processing 1 - untreated control 2 - no ATP depletion 3 - ATP depleted 4 - Phosphocreatine 5 - Creatine kinase 6 - CP + CK CG Prucca et al. Nature 456, (2008) doi: /nature07585 Next Question Question: Are the Giardia RNAi components directly involved in regulating the VSP transcripts? Methods: RNA silencing of the Dicer, RdRP and Ago components. Results: RdRP antisense resulted in ~ 75% reduction of RdRP mrna (supplemental Fig. 9) Dicer antisense resulted in ~ 65% reduction of Dicer mrna (supplemental Fig. 9) When RdRP or Dicer was silenced, trophozoites expressed more than one VSP on the cell surface.

11 Giardia RNAi - Figure 3 Expression of different VSPs in Giardia rdrp- and dicer-knockdown transgenic trophozoites. Western blot demonstrates that many VSP proteins are now present in the silenced cell lines - used antibody to the conserved C-terminal region of VSP proteins. Lanes 1 and 2 while the control cell line shows just a single VSP - 9B10. CG Prucca et al. Nature 456, (2008) doi: /nature07585 Summary of Antigenic Variation Post-transcriptional regulation Dicer Giardia Antigenic Variation Still more Questions What unresolved questions can you come up with?