Impressive paper on control of message activity during meioisis (Saccharomyces)

Here's a summary of the Journal club article I just presented at the UCSF RNA club.  It was a super fun paper to review, and volunteering to do it forced me to read both the paper itslef (which had a terrifying 100 pages of supplemental data!) and the requisite background to not mangle the paper's findings.

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!

Talk summary: human vs. viral genetic arms race

The speaker at yesterday's microbial pathogenesis series (UCSF) was exceptional.   Harmit Malik spoke about viral co-evolution with (primate) host defense responses, casting the process as a genetic arms race. Using signature gene sequences and mining available genomes, he traced HIV, hepatitis C, etc. back to various stages in primate evolution.  Because retroviruses insert their DNA into ours (sometimes becoming part of our germ line), by looking for signature viral genes one can type the viral burdens of our ancesters back in some inferred cases by hundreds of millions of years.  Harmit then went into case studies showing how a virus acquired functions to subvert host defenses and described the host response to that - all at the amino acid level of key proteins over evolutionary time.  His lab further showed that if you reversed a host mutation back to its "ancestral" state, it lost the ability to block viral activity.  The research article is unfortunately closed access, so I won't link to it.

Anyway this led to a discussion with down-the-hall colleague Suzanne Noble in which she pointed out that viruses are thought to be most virulent when they hop to a new host species, decimating it because no host-pathogen equilibrium has yet been established.  Over time, the host evolves a response and the virus becomes more stably integrated within the population.  So is the end "goal" of a (retro)virus to not be a virus at all, but to be a heritable genetic element in the host genome?  Another colleague in our lab, and always-fun-discussion partner, Mark Voorhies, pointed out that the best of all worlds is to retain the ability to synthesize virions and induce lysis, such that if the host runs into trouble the latent virus can jump ship.  Such a strategy is well-characterized in bacteriophages and is called lysogeny.  However in this case I suspect it's more complicated.  For instance, the less frequently a virus escapes its host, over evolutionary time, the less capable it will be to successfully escape.  The requisite genes, having been isolated from selective pressure while dormant and transmitted, will not necessarily be competent to escape the host. 

Anyway it was a fun morning.  If you ever get a chance to hear Harmit speak I strongly recommend taking it; we see many speakers at UCSF and he was a stand out example of how to give a talk.

If you have any comments or want to set me straight on retroviral evolutiion and its consequences, please comment away.

update: here's the PLoS Biology comment introducing Harmit's publication:

Future scientific advances and the United States

Small comment inspired by this article:

Great read. Sad that the US is falling behind, although maybe inevitable given the national arrogance that always accompanies hegemony. You start to take success and power for granted while listening to your own misleading stories of intrinsic greatness.  Aside from that, one quibble is that he's predicting future accomplishments based on what Americans/westerners have generally emphasized.  If we're diminishing as an innovative force due to loss of infrastructure and investment, then the conceptual flavor of innovation will also change as different cultures take the lead in asking the big questions.

A statement on nonviolence

This arose from an offline conversation with someone advocating - or for the purpose of debate pretending to adocate, I can't tell - for violence in civil rights movements, "Occupy Wall Street" included.  Anyway here's my response to him, which predictably comes down on the side of nonviolence but for reasons that surprised me when I ended up writing this response, edited to correct grammar and typos:

" I think the heart of my reaction boils down to believing that almost anyone has the capacity to respond with empathy when presented with a more complete description of the world's dynamics.  Even the most rabid conservatives who call for wholesale slaughter of brown people in the Middle East will respond with empathy and concern when someone they know is in trouble - even if responding with help and support violates every principle they spent years espousing or even enforcing. I call this one's "empathy radius" and use it to distinguish those who end up being  (a modern United states) conservative from those who become progressive: what is your capacity to identify with others?

I agree that credible threats of force contribute to behavioral change in others, which I take it underlies your invoking the Black Panthers and the "Arab Spring" in Egypt.  But let me ask you this: what changes in those others and what are the consequences of those changes? It's pretty well-established that strong emotions and credible threats are very effective in shutting down large portions of human brains while they resort to limbic-based reasoning. Therefore, by invoking violence credibly, you are diminishing the humanity of your "opponent." You also accept the terms of their end of the debate by acknowledging their "otherness." That may, as you indicate, result in short-term successes but does absolutely nothing to address the root cause - the willingness to see and treat others as outside of one's empathy radius. When people say that the enemy has won because you started acting like them, they may mean that you have debased youself. That's not what I hear.  I hear that "they" have won because you have done nothing to substantively advance and change how conversations about power are constructed. You are now playing their game on their terms by having accepted their propositions so fundamentally that the acceptance defines everything fundamental about your response. They're not winning by eliciting a violent response from you; they're making you into them."