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First post here, so bare with me if I violate any etiquette or formatting rules.

Let's say I've got a protein in humans. When it interacts with an ncRNA found in humans, it does something that I'm very interested in. I see a highly homologous (~85% AA identity) protein in plants, and it's widely conserved. I'm interested in eliciting the same activity of this protein in plants. Only problem is that there's no native plant-produced ncRNA to interact with my protein of interest in the same way. So I had an idea:

What if I introduce a transcript to plants that addresses this problem? If the plant version of this protein interacts with this (introduced) RNA in the same way it will be really cool. I'd like to test this hypothesis with some synthesized constructs. But before I plunk down thousands of dollars on a few constructs, I'd like to explore any predictive or analytical tools to strengthen the case. I'm aware the odds are against me.

Thanks!

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    $\begingroup$ I can't think of any predictive tools, but I think you should simplify the system as much as possible first. Instead of trying to express the ncRNA in plant, get the purified plant protein and purified ncRNA, maybe by in vitro transcription. Mix the two together and see if you can use coprecipitation or crosslinking or something to look for binding. Fluorescent anisotropy might be cool if you have a fluoremeter with polarizers, you could label the RNA with a flourophore, then measure the tumbling rate, once it binds the protein the tumbling should slow down. Use the human protein as control. $\endgroup$
    – user137
    Aug 5 '15 at 22:33
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The ncRNA sequence from human may bind to the plant protein in the plant cells, but so what? You may be able to say the plant protein works as RNA binding protein, but no more than that. Another concern is that the plant protein may not recognize the same RNA sequence to the human protein.

There are several ways to find RNA partners of RNA binding proteins.

SELEX

This method is used to find consensus sequences that your protein recognizes in vitro. Once you find consensus sequences, you can search ncRNAs from the plant.

CLIP

This is similar to Chip assay. You could find real targets of your RNA binding proteins

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  • $\begingroup$ With my experimental plan, I plan to use a reporter transcript. I will be able to say more than "it's an RNA binding protein". My primary concern is precisely that the "plant protein may not recognize the same RNA sequence to the human protein" if at all. SELEX and CHIP will likely help me in my objective here. Thank you for your suggestions. $\endgroup$ Aug 6 '15 at 2:59
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    $\begingroup$ I would be careful using SELEX to find endogenous RNAs that bind the protein, it would probably find an RNA aptamer that binds the protein well, but there is no guarantee that the resulting RNA would actually bind in a relevant manner, or just be a novel sequence that happens to bind well. Being able to make an RNA to bind almost anything is why SELEX is so useful. $\endgroup$
    – user137
    Aug 6 '15 at 12:58

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