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The use of RNAi to suppress gene function in industrial fungi Nigel S. Dunn-Coleman BMS Meeting, Manchester September , 2005. RNAi pathway in N. crassa. mRNA cleavage and degradation. AAA. QDE2. mRNA. RISC. AAA. endogene. Nucleus. siRNA. transgenes. DNA\DNA interaction. QDE3. DCR1.
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The inverted repeat is placed under the control of a
quinic acid inducible promoter
Southern Blot T. ressei transformed with N.crassa albino gene (al-1) RNAi vector
3 12 14 24 25 49 51 57 60 M B M B
Small interfering RNAs corresponding to the al-1 dsRNA.The transformants 1, 24 and 42 show a clear accumulation of siRNA. The RNA was extracted from cultures either in quinic induced (i) or non-induced conditions (ni).
The 6xw is a Neurospora silenced strain with multiple copies of transgene, used as positive control. The strains B1 and B7 are also positive controls.
Genencor in collaboration with academic researchers has developed laccase as a reporter system for gene activity for A. niger and T. reesei (submitted)
laccase gene over expressed in T. reesei strain P37(ABTS indicator plates)
UUAGGUCAURNAi hairpin construct targeting T.reesei expressed Stacchybotyris laccase B gene
repeat, 500 bp lccB
500 bp lccB sense strand
Effective suppression of
siRNA Northern 24 bp lccB biotin labeled specific probe
1. anti-probe 24 bp DNA Oligo (positive control)
2. P37 expressing laccase, base strain (negative control)
3. P37 expressing laccase, base strain (negative control)
4. P37; parent strain (negative control)
5. RNAi strain, lccB1-8 (laccase silenced)
6. RNAi strain, lccB1-21 (laccase silenced)
7. RNAi strain, lccB1-26 (laccase silenced)
8. RNAi strain, lccB2-5 (laccase silenced)
9. RNAi strain, lccB2-7 (laccase silenced)
The mutations in the cot1 gene can results in compact morphologies
areA is a positively acting regulatory gene which has been shown to be essential for activating genes encoding enzymes, permeases, needed to acquire nitrogen for the environment
areA has recently been shown in Aspergillus to play a positive role in cellulase expression
creB and creC play a role in conjunction with cre1 in the regulation of cellulases. Make RNAi versions of these genes to determine impact on cellulase expression.
The genes for all three of these regulators are found in the JGI T. reesei genome sequence
No mutants for areA, creB or creC exist in T. reesei
Probable creA mRNA degradation product
Lanes 1-7: P-37 independent cre1-RNAi transformants
Lane 8. P-37 transformed with IRal-1 (control)
Lane 9: P-37 untransformed (control)
Second demonstration that RNAi can be used to regulate morphology in T. reesei
These transformants are also carbon catabolite de-repressed
Mutations in creA, creB and creC lead to significant carbon catabolite de-repression of cellulase in A. nidulans
The role of the CREB/CREC complex is to remove ubiquitin from specific substrates
Mutants examined to-date appear to be loss of function mutations
(K Kelly et al)
Two T. reesei homologs in JGI T. reesei genome
The small, 76-residue, protein is found both as free monomer in eukaryotic cells, and co-valently attached to itself and other proteins. The C-terminus of ubiquitin forms an isopeptide bond with the amino group of a lysine side chain in a target protein. In this way proteins can be covalently modified by the addition of ubiquitin (cf. phosphorylation) which may alter the target protein's function. If a chain of multiple copies of ubiquitin is atached to a target proteins this appears to target the protein for degradation by the large intacellular protease known as the 26S proteasome. However, recent evidence suggests that ubiquitination (or ubiquitinylation - whatever you prefer!) can target proteins for other fates besides degradation by the proteasome.
Ubiquitinylation has been compared to phosphorylation (hence the change in the word) , and indeed the emeging scope and universality of this protein modification suggests this comparison is not fanciful. A great deal of interest is focusing on the multiple roles of ubiquitinylation, not just from the basic science viewpoint, but also because of its importance in disease.
Evidence of DICER
1 2 3 4 5 6 8 9 10 11 12
Line 1: Standard
Line 2: control P3-37
Line 3: Sample A2
Line 4: Sample A8
Line 5: Sample A9
Line 6: Sample A34
Line 8: control P-37
Line 9: Sample CB 9
Line 10: Sample CB 21
Line 11: Sample CB 4
Line 12: Sample CB 5
1 3 4 5 6 7 8
Line 1: Standard
Line 3: control P-37
Line 4: Sample CC1
Line 5: Sample CC5
Line 6: Sample CC53
Line 7: Sample CC19
Line 8: Sample CC 48
mRNA cleavage and degradation
These are similar results to those obtained earlier in N. crassa
Inverted repeat transgene
Summary RNAi Pathway
pIR induces higher silencing frequency than a plasmid (pX16) containing a single copy
is sufficient to induce silencing
Relative copy number of full-length pIR
The presence of a single full-length copy of pIR is sufficient to induce silencing of al-1 gene.
However, very few (less than 10%) of the transformants strains show an “inducible” silencing
IT IS IMPORTANT TO USE A VERY TIGHTLY REGULATED PROMOTER
E Forrest, G Marcino & C Cogoni
University of Rome