BMCB625:ncRNA

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BMCB625 Advanced Topics in Molecular Biology

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(Homework) Questions

Larry

  1. Q1. Does the publication of the other model showing FMRP binding directly to target mRNAs invalidate their model?
  1. Q2. Even though it is still not understood how the lack of FMRP causes such a severe clinical phenotype? Does this imply more physiological roles than presented here?

Chayne

As more of a potential discussion point:

This paper describes FMRP as being a transciptional repressor, particularly towards different cytoskeletal protein mRNAs and calcium/calmodulin kinase IIa mRNA. This seems paradoxical as the development of healthy mushroom dendritic spines would require that these proteins be upregulated, not repressed. This would suggest that patients with Fragile X syndrome should have large and strong dendritic spines due to the lack of FMRP repression. However, this is not the case. Fragile X patients have under-developed thin dendritic spines with weak synapses characterized by LTD. As an example, a large percentage of the post-synaptic density (PSD) is CaMKIIa, and mushroomed spines with strong LTP are characterized by an increase in PSD, thus an increase in CaMKIIa expression must occur. This doesn't make sense if FMRP is repressing CaMKIIa mRNA.

Is FMRP really a repressor of translation in vivo? Or could it be serving to ensure the proper localization of mRNA or some other dual functional role?


Jon

Q1: Why do you think (in regards to fig 4) that addition of antibody to brain extracts (lane 5) removes three major complexes seen in lane 4?


Q2: It is interesting the authors speculate that BC1 is tRNA(ala) like. How might one more specifically examine this interaction.


Chris

  • Q1:

The authors claim that FMRP binding to BC1 RNA is “...specific and stoichiometric since the complex can be easily competed off by 100-fold excess unlabeled BC1 RNA...” (Fig 4, Ln3, versus Ln6&7)

I agree it demonstrates specificity, but how is this stoichiometric?

  • Q2:

Figure 6: This is a good assay to demonstrate base-pairing interactions between BC1 and a variety of mRNA’s, but how could we demonstrate this is occurring in vivo?



Mahta

Q1. From the data can you envision a model in which several RNAs similar to BC1 act as "bridges" or "adaptors" between FMRP and mRNAs targetd for degradation?

Q2. High salt is needed to efficiently immunoprecipitate FMRP (the authors state that the C-terminal can be masked at physiological concentrations). Also, the recombinant FMRP is only stable in 1M salt. Is this troublesome?


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