Question
Describe an example in which an miRNA activates translation of a gene. How was this activation assayed? Present evidence that base-pairing between this miRNA and the mRNA's ARE is important in activation.
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One well-studied example is miR-21, which has been shown to enhance the translation of certain target mRNAs, including those involved in cell proliferation and survival. Show more…
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You are interested in the newly identified iGENE signaling pathway and performed an RNA-seq experiment to identify candidate target genes that are regulated by iGENE. To do so, you overexpressed the iGENE cDNA under control of the CMV promoter in HEK293 cells and sequenced mRNA transcripts from transfected and untransfected control cells. A bioinformatic comparison revealed that 1,236 genes were upregulated, while 582 genes were downregulated when the iGENE is overexpressed. Among the upregulated genes, iTARGET was expressed 16 times higher in transfected than in control cells. As a next step, you now want to understand whether iTARGET is a direct target of the iGENE protein. List three different ways how you could demonstrate a direct interaction between the iGENE protein and the iTARGET gene promoter. Describe one approach in detail. How could the same iGENE protein upregulate the expression of some genes and downregulate the expression of others?
By searching a human genomic database, you have found a gene that encodes a protein with weak homology to Argonaute, a factor present in complexes that bind to certain miRNAs and mediate their ability to regulate the stability or translation of target mRNAs (review Figs. 17.32 and 17.33 ). a. How would you determine which specific miRNAs might be associated with the new protein you discovered? (Think about how you might use a variation of the ChIP-Seq technique described in Fig. 17.18 to explore this question.) b. If a mouse could be obtained that was homozygous for a null mutation of a gene almost identical to the human gene you found, how could you use this mutant mouse to ask what mRNAs might be targeted by the miRNA-RISC complexes containing your Argonaute-like protein?
Andrew Fire, Craig Mello, and their colleagues were among the first to examine the effects of doublestranded RNA on gene expression (A. Fire et al. 1998 Nature $391: 806-811$ ). In one experiment, they used a transgenic strain of $C$ elegans into which a gene $(g f p)$ for a green fluorescent pigment had been introduced. They injected some worms with double-stranded RNA complementary to coding sequences of the $g f p$ gene and injected other worms with double-stranded RNA complementary to the coding region of a different gene $(u n c 22 A)$ that encodes a muscle protein. The photographs on the next page show larvae and adult progeny of the injected worms. Green fluorescent pigment appears as bright spots in the photographs. a. Explain these results. b. Fire and Mello conducted another experiment in which they injected double-stranded RNA complementary to the introns and promoter sequences of the $g f p$ gene. What results would you expect with this experiment? Explain your answer.
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