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Question: problem 4 micrornas are noncoding rnas 2024 nt in size...

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PROBLEM 4 microRNAs are non-coding RNAs 20-24 nt in size. They play a key role in posttranscriptional regulation of eukaryotic gene expression. It is known that they bind to proteins of the ARGONAUTE (AGO) family, and that they use base pairing to recruit the AGO-miRNA complex to partially complementary mRNA, often referred to as “target mRNA”. Recruitment of AGO can bring about translational repression of the target mRNA.
To study how miRNAs repress translation of target mRNA, a Drosophila embryonic cell culture was transfected with plasmids encoding Renilla Luciferase (RL) either without miRNA target sites in the 3’Untranslated Region (Con) or with three sites partially complementary to the miRNA let-7 (3xBulge; Figure 4-1). The cells used in these experiments express let-7 endogenously. The plasmids also encode a nontargeted Firefly Luciferase whose light emission upon substrate conversion can be distinguished from that of Renilla Luciferase.

3UTR RL or FL Con No sites Figure 4-1 Schematic representation of reporter constructs used to monitor miRNA dependent inhibition of gene expression The researchers first wished to establish that their reporter mRNA really is subjected to let-7 guided repression of mRNA translation. Relative Renilla Luciferase activity was measured in total cell lysates transfected with either Con or 3xBulge plasmids. The results are shown in Figure 4-2 15 AL 0.5 Figure 4-2 Relative Renilla Luciferase activity measured in total cell lysates upon transfection with Con and 3xBulge plasmids.

Question 4-1 Why is it necessary to include a non-targeted Firefly Luciferase in these experiments, and what is meant by “relative Renilla Luciferase activity”?

Question 4-2 Can you conclude from the results shown in Figure 4-2 that let- 7 represses RL-3xBulge expression?

Next, a polysome profiling experiment was performed by ultracentrifugation of total cell lysates through a 10-50% sucrose gradient. The lysates were prepared from cells transfected with either Con or 3xBulge plasmids. Fractions across the gradient were collected, and the absorbance at 260 nm was monitored during fraction collection. Northern blot analyses were used to quantify the presence of RL (Con or 3xBulge) and β-actin mRNAs across the gradients. The results of these experiments are shown in Figure 4-3.

Question 4-3 Which biological molecules account for most of the absorbance measured at 260 nm?

Question 4-4 Compare the profiles obtained from cells transfected with Con and 3xbulge plasmids.

Question 4-5 Why is β-actin mRNA abundance monitored across the gradient, and what do the β-actin northerns show?

Question 4-6 Describe the distribution of RL(Con) and RL(3xBulge) mRNA across the gradient.

The drug puromycin imitates a charged tRNA and is able to bind to the A-site of ribosomes. This leads to ribosome drop-off from mRNA following formation and release of a puromycylated nascent polypeptide chain.

Question 4-7 Draw an outline of how the A260 profile would look if the cells had been treated with puromycin prior to lysis.

Question 4-8 Use the data shown in Figure 4-3 to argue what can be concluded on the mechanism used by let-7 to repress translation of target mRNA. What step of the translation mechanism is affected? Explain your answer.

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