THE EXOSOME REGULATES

CIRCADIAN GENE

EXPRESSION IN A

POSTTRANSCRIPTIONAL

NEGATIVE FEEDBACK LOOP

Today’s Paper:

Lecturer: Kalesh Sasidharan Date: 2009 October 26

TITLE

Keywords:

1. Exosome

2. Circadian Gene Expression

3. Posttranscriptional Regulation

4. Negative Feedback Loop

The exosome complex is a multi-protein complex capable ofdegrading various types of RNAmolecules.

The core of the exosome containsa six-membered ring structure towhich other proteins areattached.

Exosome complexes are found inboth eukaryotic cells andarchaea, while in bacteria asimpler complex called thedegradosome carries out similarfunctions.

Source: http://en.wikipedia.org/wiki/Exosome_complex

A circadian rhythm is a roughly-24-hour cycle in thebiochemical, physiological or behavioral processes of livingentities, including plants, animals, fungi and cyanobacteria.

Circadian rhythms are endogenously generated, and can beentrained by external cues, called zeitgebers, the primaryone of which is daylight. These rhythms allow organisms toanticipate and prepare for precise and regularenvironmental changes.

Source: http://en.wikipedia.org/wiki/Circadian_rhythm

SUMMARY

Keywords:

1. Neurospora

2. FRQ and FRH

3. FFC complex

4. WHITE COLLAR complex

5. Clock-Controlled Genes

NEUROSPORA CRASSA

Neurospora crassa is atype of red bread mold ofthe phylum Ascomycota.

N. crassa is used as amodel organism because itis easy to grow and has ahaploid life cycle thatmakes genetic analysissimple since recessivetraits will show up in theoffspring.

Its entire genome of sevenchromosomes has beensequenced.

Source: http://en.wikipedia.org/wiki/Neurospora_crassa

http://www.nih.gov/science/models/neurospora/

http://www.apsnet.org/online/feature/view.aspx?ID=326

FRQ, FRH AND HELICASE

The Neurospora circadian

system is built around a cellular

oscillator protein called

"frequency" (FRQ) whose

expression fluctuates with the 24

hour light-dark cycle.

FRH - FRQ-interacting RNA

helicase (FRH)

Helicases are a class of enzymes

vital to all living organisms.

They are motor proteins that

move directionally along a

nucleic acid phosphodiester

backbone, separating two

annealed nucleic acid strands

(i.e. DNA, RNA, or RNA-DNA

hybrid) using energy derived

from ATP hydrolysis.

Source: http://en.wikipedia.org/wiki/Helicase

http://www.ambion.com/techlib/hottopics/frq_04182003.html

BACKGROUND

Source: https://www1.qiagen.com/GeneGlobe/PathwayView.aspx?pathwayID=114

WHAT WE CAN IMAGINE FROM SUMMARY AND BACKGROUND!!

FRQ FRH

frq

WC1WC2

frq RNA

FRQ

FRQ

FRH

FFC

Exosome

With

rrp44

rrp44

WC1WC2

Rrp44 Protein

Post-transcriptional

regulation

Controls circadian output

Through Clock-Controlled Genes

FRQ

FRQ

FRH

FFC

FIGURE 1

B

C

INVERT-REPEAT WITH QUINIC ACID

Without Quinic acid = wt

frh Promoter frh

qa Promoter frh invert repeat

frh RNA

FRQ

With Quinic acid = frhΔ

frh Promoter frh

qa Promoter frh invert repeat

frhRNA

frh RNA

FRQ

CHROMATIN IMMUNOPRECIPITATION

(CHIP) ASSAY

Source: http://www.nature.com/nprot/journal/v3/n6/images/nprot.2008.68-F1.jpg

FIGURE 2

Thiolutin is a sulfur-containing

antibiotic, which is a potent inhibitor

of bacterial and yeast RNA

polymerases.Source: http://en.wikipedia.org/wiki/Thiolutin

RNASE H ASSAY

RNase H specifically degrades the RNA inRNA:DNA hybrids and will not degrade DNA orunhybridized RNA.

It is commonly used to destroy the RNA templateafter first-strand complementary DNA (cDNA)synthesis by reverse transcription, as well asprocedures such as nuclease protection assays.

RNase H can also be used to degrade specificRNA strands when the cDNA oligo is hybridized,such as the removal of the poly(A) tail frommRNA hybridized to oligo(dT), or the destructionof a chosen non-coding RNA inside or outside theliving cell.

Source: http://en.wikipedia.org/wiki/RNase_H

RNASE H ASSAY

RNA

mRNA

frq mRNA Poly(A) tailfrq 3’ UTR

Longer Poly(A) tail

frq mRNA Poly(A) tailfrq 3’ UTR

Upstream frq 3’ UTR

complimentary DNA

frq mRNA Poly(A) tailfrq 3’ UTR

Upstream frq 3’ UTR

complimentary DNA

RNase H

frq mRNA Poly(A) tailfrq 3’ UTR

Upstream frq 3’ UTR

complimentary DNA

Oligo d(T)

frq mRNA Poly(A) tailfrq 3’ UTR

Upstream frq 3’ UTR

complimentary DNA

Oligo d(T)

RNase H RNase H

frq mRNA Poly(A) tailfrq 3’ UTR

Poly(A) tail frq 3’ UTR

frq mRNA

FIGURE 3

FIGURE 4

RACE TUBE ASSAY

A race tube is a long glass tube (28 inch long) that isbent up at both ends to hold an agar medium. Myceliaor conidia are inoculated at one end of the race tubeand the fungus grows toward the other end.

To synchronize the cells to the same time of day, thecultures are germinated in constant light for a dayand then transferred to constant darkness. Underthese conditions, Neurospora initiates asexualdevelopment about every 22 hrs (Loros and Dunlap2001).

As a result of asexual development, Neurosporaproduces orange colored spores creating the “banding”phenotype. This easily tractable clock phenotype hasbeen integral in dissecting the genetic structure of theNeurospora circadian oscillator.

Source: http://www.fgsc.net/fgn51/fgn51park.htm

RACE TUBE ASSAY

Source: http://www.fgsc.net/fgn51/fgn51park.htm

http://www.biochemsoctrans.org/bst/033/0949/bst0330949f01.htm?resolution=STD

FIGURE 5

FIGURE 6

FIGURE 7

FIGURE 7 - MODEL

http://customersrock.files.wordpress.com/2007/04/thank-you.jpg