Giardia RNAi Paper Discussion. Supplemental - VSG clonality. Variant-specific surface protein VSP9B10

Transcription

1 Giardia RNAi Paper Discussion Supplemental - VSG clonality VSP9B10 Green - VSP9B10 antibody Blue - DAPI Demonstration that cell line expresses a single VSP Variant-specific surface protein Outside Inside VSPs are Cysteine-rich % cysteine (disulfide linkages) CXXC motifs usually associated with DNA binding proteins VSP repertoire: possible variants ~190 expressed (reported) Variable N- terminus, Conserved C-terminus. Possibly all VSP genes are transcribed, but ONLY 1 protein is stable. VSP switches every 5-13 generations

2 First Question Question: Which VSPs are transcribed? Methods: Nuclear Run-on vs. Northern blotting 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?

3 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 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) characteristic of dsrna mediated reactions RdRP Activity Dicer Argonaute (Ago) The three RNAi components are constitutively expressed in both life cycle stages.

4 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. 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 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. 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.

5 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. Summary of Antigenic Variation Post-transcriptional regulation Dicer Giardia Antigenic Variation Still more Questions What unresolved questions can you come up with?