Link to article, for those with access: http://www.sciencemag.org/content/335/6068/552.abstract
I'll have more to say about what was reported in this paper - which I consider a landmark piece of work - as well as a layperson's summary...later. Right now I gotta head over to the Fischbach lab and run some preparative HPLC. Super exciting day.
Below are my notes for the presentation, for the aficionados who want to scan them. I was going to upload my slides, but they contain images from this and other non-open-access works. Not sure it's OK for me to present them here without the express permission of their authors and publishers (the latter, of course, sucks).
Brar et al (2012) JC presentation
Introduction
1. Lots of data in paper
Not going to be able to get through all biological and technical developments in 1 hour
Will focus on interesting biological questions answered
With technical review necessary to understand results
And biological review of context
Cell cycle control
translational control
Ambitious project yielding high-resolution data set that authors will be able to explore over several careers
2. Meiosis
Specialized developmental process yielding haploid germ cells from diploid progenitors
Chromosomal DNA is compacted by factos up to 1:10,000 relative to vegetative state
Transcription globally silenced
TFs and histones have been shown to exchange into/out of chromosomes
Mammalian mitosis: RNA Pol II excluded from mitotic chromosomes (Parsons & Spencer MCB 1997)
How is highly coordinated, precisely-timed process regulated?
Majority of DNA is tightly packaged and less accessible.
What role does translational regulation play during meiosis?
Authors wanted unbiased, high-resolution view of transcription and translation during cycle
3. History of relevant systematic approaches
1998: Ira Herskowitz (along with Joe DeRisi, Michael Eisen and Pat Brown) inventory changes in message levels
through meiosis in yeast. Develop basic structure echoed in current paper:
=> identify stages cytologically
=> Cluster genes by expression pattern
=> relate to cytological stage
=> use functional annotation to predict and mutagenesis to validate
2003: Current co-author Ghaemmaghami (here at UCSF with O'shea and Weissman) attempts to define
global protein expression in yeast.
2003: Pat Brown and Dan Herschlag labs collaborate to assess # active ribosomes assoc with all transcripts
microarray based
had to physically bin polysome peaks, leading to steps in data
2009: Landmark paper from Weissman lab. Used RNAseq to define ribosome footprint of each transcript.
Now able to precisely quantify # ribosomes per transcript, # transcripts and ribosome position on transcript
"Ribosome profiling"
2012: Using ribosome profiling approach, Weissman lab returns to 1998 Herskowitz project
Goal: precisely define transcriptional and translational activity using RNAseq sensitivity
Mitosis/meiosis review
Allows sexual reproduction, genetic recombination
In yeast, gametogenesis (sporulation) -> sexual spores
Two distinct processes: meiosis and cytokinesis
Meiosis: faithful replication of parental DNA, controlled recombination (crossover), separation 2N -> 4N -> 1N
Cytokinesis: physical separation of cells
Highly regulated, precise timing
A. Review meiosis in yeast
1. Overall process (basic)
2. Features specific to Sc
Induced by lack of glucose and nitrogen, + nonfermentable carbon source
IME1 = master regulator
NDT80 induces, requires Ime2 kinase (and Ndt80 accumulation) for activation
Ime2 phosphorylates Ime1 => degradation and Ndt80 => activation of meiotic progression
(Sopko et al MCB 2002)
3. Meiosis in Sc
Pre-meiotic DNA replication (S phase) resembles pre-mitotic in many ways
but is slower and requires different factors like MUM2.
Passage through meiotic S-phase required for DSBs, meiotic recomb and Synaptonemal complex formation
Pachytene checkpoint: after DSB/crossover => is damage repaired?
Questions:
How is segregation of organelles regulated? When does segregation happen?
Check claim that clustering of genes recapitulates cycle phasing
review staging controls used
good review of uORF regulation/activity
why didn't authors co-IP ribosomes?
review mitotic checkpoints to explain YDR506 and YLR445W experiments
how did they differentiate between protection by ribosome and protection by secondary structure, stress granules, etc?
monosome cut (RNase I treated vs. ctrl) then resolved by gel and excised 28 bp region.
Methods
1. RNA prep
+CHX: Harvest cells by filtration; drip into liquid nitrogen; pulverize
Take total RNA aliquot, treat w/ RNAse I vs U/T ctrl
sucrose gradient, monosome cut, excise 28bp region from polyacrylamide gel
add poly-A tail to 3' end
ss cDNA amplification; circularize; cut with ApeI; now do PCR to amplify from both ends
2. RNA sequence mapping
seq = variable mRNA read + polyA sequence
Align w/ up to 3 mismatches
If aligned seq matches region with AAA... then ambiguous
Resolved by using shortest and longest fragment w/ highest score
Degenerate reads (>= 18 of first 22 nucleotides were ’A’ bases) were eliminated.
rRNA hits were discarded
(Ingolia 2009; in present paper rRNA sequences experimentally subtracted)
Align all reads independently against:
yeast genomic sequences,
yeast processed protein-coding genes
yeast processed non-coding RNAs
if no alignment, then:
processed protein-coding transcripts
Then filter out low-complexity mappings:
discard >2 mismatches, <18 bp alignment against genome
Start codons were called by:
AUG and "initiation context score" >0.001
non-AUG (1 bp difference) and "initiation context score" >0.01
context score came from sequence matrix derived from AUG region of highly expressed genes
Ribosome density correlates better w/ protein abundance than does mRNA abundance (Ingolia et al 2009)
More later!
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