Resolving the dynamics of gene action at atomic through organismal scales

http://dordognesnicest.com/the-cave-of-rouffignac/ ≈12000 BCE

Sequencing mastodon and woolly mammoth genomes

Central Dogma of Molecular Biology

• DNA makes RNA makes protein – DNA -> DNA is replication

• DNA bases (4 standard): G, A, T, C

– DNA -> RNA is transcription • RNA bases (4 standard) : G, A, U, C

– RNA -> protein is translation • Protein amino acids (20 standard):

A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y

• Modified dogma – RNA -> DNA is reverse transcription

Oscar Miller (1925-2012)

Miller, O.L., Jr., Hamkalo, B.A., and Thomas, C.A., Jr.

(1970). Science 169, 392-395.

RNA Pol

Visualization of bacterial genes in action

Double-strand DNA

Single-strand DNA

Translation of DNA – 3 forward

reading frames

Translation of DNA –

first reading frame

IFIHAVESEENFXRTHERITISXYSTANDINGXNTHESHXXLDERSXFGIANTS

Sir Isaac Newton to Robert Hooke 5 Feb 1675

http://digitallibrary.hsp.org/index.php/Detail/Object/Show/object_id/9285

IF I HAVE SEEN FURTHER IT IS BY

STANDING ON THE SHOULDERS OF GIANTS

http://www.nature.com/nature/journal/v456/n7218/extref/nature07517-s1.pdf

Figure S1. a. Structure of the

reversible terminator 3’-O-

azidomethyl 2’-deoxythymine

triphosphate (T) labelled with

a removable fluorophore. b.

Structure of the incorporated

nucleotide after removal of

the fluorophore and

terminator group. Each of the

four nucleotides have an

equivalent structure to the

one shown here, except for

the different base and a

corresponding base-specific

fluor.

http://array.mc.vanderbilt.edu/Sequencing_slides-Intro_Seminar.pdf

http://www.nature.com/nature/journal/v456/n7218/extref/nature07517-s1.pdf

http://www.nature.com/nature/journal/v456/n7218/extref/nature07517-s1.pdf

http://www.nature.com/nature/journal/v456/n7218/extref/nature07517-s1.pdf

http://array.mc.vanderbilt.edu/Sequencing_slides-Intro_Seminar.pdf

Fig.1 Cycles 9 through 13 of stack 87 for the variant MFN_R364W. Colors are

red: C, green: G, blue: T. The expected sequence is TCTGG (blue, red, blue,

green, green). The location of the variant is circled.

http://www.openwetware.org/wiki/User:Andrea_Loehr

Fig.2 Cycles 11 through 15 of stack 27 for the control sequence for

variant MFN_R364W. Colors are red: C, green: G, blue: T. The expected

sequence is TCCGG (blue, red, red, green, green). The location of the

control is circled.

http://www.openwetware.org/wiki/User:Andrea_Loehr

Next Steps

Next, we will automate the image processing with a shell script and

ImageMagick. We will install and run the Swift software on BOINC.

http://www.openwetware.org/wiki/User:Andrea_Loehr

Contact Info

Andrea Loehr

Harvard-Smithsonian Center for Astrophysics

Cambridge, MA, USA

Email me through OpenWetWare

I work at the Harvard Smithsonian Center for

Astrophysics. I learned about OpenWetWare through the

Church Lab, and I've joined because I have started to

work on a project with the Personal Genome Project

(PGP). Our goal is to set up a volunteer computing project

PersonalGenomes@Home, analogous to seti@home.

http://www.openwetware.org/wiki/User:Andrea_Loehr

Levels of genetic control

• Chromatin structure

• Transcriptional control

• Post-transcriptional control

– RNA capping, intron processing, editing, polyadenylation

– RNA export

– RNA stability

• Translational control

• Post-translational control

– Protein modification, splicing, stability

Translational control

• Global control

– Changes in the translational machinery or masking of mRNA

• mRNA specific control

– Cis-acting sequences affecting translation

• Binding to factors

• Binding to miRNA

• Effects on ribosome behavior at the level of initiation, elongation or termination of translation

Galagan et al. 2003 Nature 422, 859-868

Growth of y14, wild-type and upf1 strains –

DELETED THE MOVIE ON THIS SLIDE

Common and rare mRNA structures

Prevalence of eukaryotic upstream open reading frames (uORFs)

• Estimates are that 15-53% of mRNAs contain uORFs • Large numbers of fungal mRNAs contain upstream

AUGs (Galagan et al., Nature 2005) – Aspergillus nidulans

• 21% EST 5’UTRs – Neurospora crassa

• 22% EST 5’UTRs

Nascent peptide control of ribosome movement

• Arg-specific ribosome stalling mediated by the nascent AAP (arginine attenuator peptide)

• AAP-mediated ribosome stalling governs mRNA stability through the nonsense-mediated mRNA decay (NMD) pathway

Origins of studies on arginine metabolism in Neurospora

AAP-mediated stalling at the uORF termination codon blocks access to the downstream initiation codon

The full-length AAP (AAP24) stalls ribosomes

with uORF the stop codon in the A-site of the

ribosome peptidyl transferase center (PTC)

Evolutionary

conservation of

the AAP

Evolutionary conservation of the AAP

Oscar Miller (1925-2012)

Miller, O.L., Jr., Hamkalo, B.A., and Thomas, C.A., Jr.

(1970). Science 169, 392-395.

RNA Pol

Visualization of bacterial genes in action

Eukaryotic mRNA translation

Eukaryotic mRNA translation

Afonina, Z.A., et al. (2014). Nucleic Acids Res 42, 9461-9469

Neurospora ribosome structure – Sachs and Beckmann labs

Eukaryotic translation initiation

-6 +4 +1 -1

-6 +4 +1 -1

Eukaryotic initiation from near-cognate codons

AAAn

GCCACCAUGG

CUGG

UUGG

GUGG

AAGG

ACGG

AGGG

AUAG

AUCG

AAAG

40S

eIF4F

eIF2

Met

Initiation codon Mammalian efficiency (%)

AUG 100.0

CUG 19.5

GUG 9.2

ACG 6.6

AUA 3.3

AUU 3.2

UUG 1.9

AUC 1.7

AAG 0.2

AGG 0.1 Ivanov, I.P., Loughran, G., Sachs, M.S., and Atkins, J.F.

(2010). Proc Natl Acad Sci U S A 107, 18056.

Determining the hierarchy of near-cognate codons in N. crassa in vivo

Wei, J., Wu, C., and Sachs, M. S. (2013) J. Biol. Chem. 288, 9549-9562

CUG and GUG are the best near-cognate codons in vivo

Wei, J., Wu, C., and Sachs, M. S. (2013) J. Biol. Chem. 288, 9549.

Wei, J., Wu, C., and Sachs, M. S. (2013) J. Biol. Chem. 288, 9549-9562

Prevalence and relevance of

potential initiation at near-cognate

codons in N. crassa

CPC1

Pezizomycotina cpc-1 homologs all share a

conserved N-terminal extension with a near cognate

start codon in an optimal context

N. crassa cpc-1 (NCU04050) –

the homolog of S. cerevisiae GCN4

Conservation of cpc-1 5’UTR coding capacity in Basidiomycota

Ribosome

profiling

Ribosome profiling: Ingolia and Weissman

Ingolia NT, Brar GA, Rouskin S, McGeachy AM, & Weissman JS (2012) The ribosome

profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-

protected mRNA fragments. Nat Protoc 7(8):1534-1550

Ribosome

profiling

Ribosome profiling: eIF1

1st nt in frame 1 1st nt in frame 2 1st nt in frame 3

Ribosome profiling: cpc-1

Genetic variation

IFIHAVESEENFXRTHERITISXYSTANDINGXNTHESHXXLDERSXFGIANTS

IFIHAVESEENFXRTHERITISXYSTANDINGXNTHESHXXLDERSXF--ANTS

Fungi that control animal behavior

http://smithsonianscience.si.edu/wordpress/wp-content/uploads/2012/08/modern-day-ant2.jpg

Ophlocordyceps takes over the ant to create a zombie ant

Analyses of gene expression patterns in fungus and ant zombie to understand gene action

de Bekker, C., Ohm, R.A., Loreto, R.G., Sebastian, A., Albert, I., Merrow, M.,

Brachmann, A., and Hughes, D.P. (2015). Gene expression during zombie ant

biting behavior reflects the complexity underlying fungal parasitic behavioral

manipulation. BMC Genomics 16, 620.

Zhai, B., Wu, C., Wang, L., Sachs, M.S., and Lin, X. (2012). The antidepressant

sertraline provides a promising therapeutic option for neurotropic cryptococcal

infections. Antimicrob Agents Chemother 56, 3758-3766.