Tag Archives: RNAi

Friday SNPpets

Welcome to our Friday feature link collection: SNPpets. During the week we come across a lot of links and reads that we think are interesting, but don’t make it to a blog post. Here they are for your enjoyment…

  • Nice video on RNA interference by Nature Reviews Genetics. You can access all of the featured RNAi multimedia links from this page, or go straight to the video on this page. [Jennifer]
  • Interesting, The Repertoire 10K (R10K) Project: RT @deannachurch: CG: go to http://t.co/rekf2Gkd for more information on joining the project! #AGBT [Mary]
  • And it’s not in the papers anymore… RT @genome_gov: Pachter: “My worst nightmare: the curse of deep sequencing” aka too much data. #AGBT [Mary]
  • Read a Nature Outlook on allergies from Nov. 2011 – lot of new philosophies & theories that I wasn’t aware of. Currently free full access is available to the Nature Allergy Outlook [Jennifer]
  • RT @andrewsu: Word cloud of NAR 2012 Database issue abstracts via http://t.co/TMtefZ0k http://t.co/2zRzZkEG [Mary]
  • Cool new option for PDB submissions: Volunteer Structures For Foldit [Jennifer]
  • RT @LouWoodley NYC tweeps – the next Science Online NYC is on March 20th on keeping the research record straight http://bit.ly/xwziUb #sonyc [Jennifer]
  • RT @GeneSherpas: “@GeneticsUpdate: Can You Be Fired for Your Genes?  http://t.co/fDaOriU” Hopefully our future doesn’t come down to this!” [Mary]
  • Ha! That was unexpected… RT @edyong209: Bizarre SNP study on genetics of choral singing. Abstract takes surprising turn in final lines. http://t.co/VkSU4fd0 [Mary]
  • RT @jacksonlab: Facing a rare #genetic disease together, the Wentzell family doesn’t let anything slow them down. #raredisease http://t.co/bscdUXoN [Mary]

Tip of the Week: MitoCheck, a human functional genomics database

As much as I love computational aspects of biology, there are times when the sort of flat and binary nature of the discipline leaves me craving some more three-dimensional,  real live cellular work.  My background was in cell biology and microtubule-associated proteins before I moved to the computational side of biology.  And there are days when I would love to see more linkage between the digital and the dimensional.  And days when I’d love to look around in the scope at mitosis and mitotic spindles again.

Today I saw it.  And I’m going to show you where.  We’ll be looking at the MitoCheck database.  Below I’ll offer some discussion of the associated research papers, and in the movie I’ll show you how to navigate around the MitoCheck site a bit to find their data online.

It was actually coverage on the BBC* that tipped me off to this resource.  And then I went looking for more.  A press release on the work provided details and links.  And then the Nature News article added additional information.

In short, this group of researchers used a couple of different genomics approaches to examine what happens to HeLa cells when you mess with the mitotic apparatus and processes.  They transform cells with either RNA interference constructs, or GFP-tagged proteins, and film what happens to the cells over time.  They analyze the movies, and make all this data available in the MitoCheck resource.  As we say here in Boston–this is wicked cool.

But now, on to the papers:  these researchers have 2 articles out that talk about the work, one focused more on the RNAi approach, and a separate one on the tagged proteins.  I’ll address them separately below.

RNA interference experiments:

In this series of experiments, the MitoCheck team started with over 20,000 protein coding genes in humans, transformed HeLa cells with the siRNAs, and let the cells divide over a couple of days.  The nuclei of the cells could be illuminated by a GFP-histone protein that they had already placed in the cells.  They could light up the cells and film them, and monitor whether cell division looked normal or not.  They were able to identify a number of cases where things were going awry.  And they were going wrong in various ways.  Sometimes there was cell death.  Other times they could see a variety of phenotypes such as delayed mitosis, binuclear, poly-lobed, or “grape” looking aberrations.   Some cells were too large.  These could all be categorized, and compared, quantified, and are now stored as movies, processed data, and phenotypic assignment in the MitoCheck database.

I have some minor concerns about how knocked-down the transcripts are–they say that the values of the target mRNAs drop a lot, but these numbers vary quite a bit (the amount of supplemental data with the paper is excruciating….).  It’s also hard to be sure what that means for the protein levels at this point.  Also, HeLa cells have some characteristics that may not be average.  But that said–as a general method and a hunting license to find genes to assess in more detail, I think this is a very excellent strategy.  If I was still in the lab, I’d try the same thing with the cell system I used to study: C2C12 cells for muscle development.  You could track whether cell fusion and myotube formation was disrupted….man, sometimes I do crave the lab still….

Tagged protein experiments:

In a second paper from the research teams, they use a similar strategy of monitoring the behavior of cells during mitosis via movies.  But this time instead of knocking down a gene, they put a GFP tag on some selected proteins (mostly mouse proteins) that they put into the HeLa cells. These are stably-transfected tagged proteins on BACs, and they call this BAC TransgeneOmics (ahem, another -omics?).  They look for where these proteins end up in dividing cells.  Again, they have movies of this available now in their database. They also pull down protein complexes and look at them in more detail with other techniques.

Again, I have minor questions about the approach: mouse proteins in HeLa cells, and the bulky GFP tag affecting interactions, expression levels, etc.  But again, as a hunting-license sort of effort, I think this is a very neat way to move downstream from digital genomics to real cells.  And it’s worth it.  The team demonstrates that you can begin to characterize the functions of unknown proteins with this strategy.

So, for this week’s tip of the week I show you MitoCheck.  I’ll show how to access this data so you can take it further if you like.  One technical note: I did have all of the issues that they talked about in their “troubleshooting” document (PDF) on my Windows machine.  I had to do all 4 of the things they recommend in there to get the movies to run. FYI.

MitoCheck site: http://mitocheck.org/

Nature paper with RNAi data:
Neumann, B., Walter, T., Hériché, J., Bulkescher, J., Erfle, H., Conrad, C., Rogers, P., Poser, I., Held, M., Liebel, U., Cetin, C., Sieckmann, F., Pau, G., Kabbe, R., Wünsche, A., Satagopam, V., Schmitz, M., Chapuis, C., Gerlich, D., Schneider, R., Eils, R., Huber, W., Peters, J., Hyman, A., Durbin, R., Pepperkok, R., & Ellenberg, J. (2010). Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes Nature, 464 (7289), 721-727 DOI: 10.1038/nature08869

Sciencexpress article with protein and complexes data:
Hutchins, J., Toyoda, Y., Hegemann, B., Poser, I., Heriche, J., Sykora, M., Augsburg, M., Hudecz, O., Buschhorn, B., Bulkescher, J., Conrad, C., Comartin, D., Schleiffer, A., Sarov, M., Pozniakovsky, A., Slabicki, M., Schloissnig, S., Steinmacher, I., Leuschner, M., Ssykor, A., Lawo, S., Pelletier, L., Stark, H., Nasmyth, K., Ellenberg, J., Durbin, R., Buchholz, F., Mechtler, K., Hyman, A., & Peters, J. (2010). Systematic Localization and Purification of Human Protein Complexes Identifies Chromosome Segregation Proteins Science DOI: 10.1126/science.1181348

*Tip of the hat to Alex who heard the BBC story and told me about it.  I owe you a cider.

RNAi to save the bees?

bee_graphicSo the other day on a political board, actually, I heard about a treatment that may be helping to fight Colony Collapse Disorder (CCD) in bees.  It had originally been posted by Treehugger.  So the blogs have been abuzz with the news.  It seems there is an Israeli company called Beeologics that has developed a product called Remembee which can knock down the IAPV (Israeli acute paralysis virus) that is purported to be one of the contributors to CCD.

The technique involves RNAi, RNA interference.  Now, this is the same technology that many environmentalists decry wildly in their Hawaiian papayas.  It was the same biotechnology that was awared the Nobel Prize a few years back.  If you aren’t familiar with it there’s a reasonable intro diagram on the Nobel Prize site that covers it.  Slide 3 and 4 in that graphic are helpful.

There’s a fairly goofy YouTube interview about the company and their efforts:

But that wasn’t enough for me, I wanted to read a paper about this.  And PubMed to the rescue, I found a paper that appears to describe what they are doing.

According the the paper they pulled out some sequences for the IAPV and for honeybees.  They also did a GFP control for a sequence unrelated to bees or IAPV.  They are required by the EPA to demonstrate that the sequences they will use to interfere with the RNA of the virus didn’t match the bee genome to elimate off-target effects.  They did this with BLAST.

They inoculate control and experimental colonies with virus.  They generated dsRNA that they fed to bees.  The bees who get the IAPV infection + dsRNA IAPV have mortality curves that are slightly lower but parallel to the untreated bees.  Those that got IAPV + GFP or IAPV only showed much steeper population declines.  Seemed pretty straightforward: feeding the dsRNA to bees was protective.

They acknowledge that IAPV may not be the only component of CCD, but that even improving the health of bee colony may help them defeat other stresses too.

I haven’t found any subsequent papers on the field trials yet, that would be great to see.  But it looks like they are definitely in the field with this.  And I’m delighted to see the environmentalists appreciate the use of biotechnology.  Gives me a warm, buzzy feeling :)

Maori, E., Paldi, N., Shafir, S., Kalev, H., Tsur, E., Glick, E., & Sela, I. (2009). IAPV, a bee-affecting virus associated with Colony Collapse Disorder can be silenced by dsRNA ingestion Insect Molecular Biology, 18 (1), 55-60 DOI: 10.1111/j.1365-2583.2009.00847.x