Question

1. We’ve now discussed instances at different times in development when the same signaling pathway is activated, but its activation results in different outcomes in terms of cell fate (i.e. gene expression) at these different times. How do you imagine this is possible?

2. We talked about lin-4 miRNA-mediated translational repression as a mechanism for temporal control of lin-14 protein expression. What components would be needed, and how would they be expressed, to achieve the same via transcriptional control? Do you see an advantage of miRNA-mediated control over transcriptional control in this case?

Answer 1

answer a) The signalling and expression of gene is highly regulated process. Not every signalling lead to expression of every gene neither expression of every gene lead same amount of gene product. This all happen because of specific regulation for every gene. There are chromatin remodeling complexes , promoters , enhancers insulators at transcription level controlling the differential gene expression. miRNA, SiRNA and various spliceosome action also been associated with regulation of gene expression thus ultimate leading to different cell outcome.

Answer b)

miRNA biogenesis starts with the processing of RNA polymerase II/III transcripts post- or co-transcriptionally. About half of all currently identified miRNAs are intragenic and processed mostly from introns and relatively few exons of protein coding genes, while the remaining are intergenic, transcribed independently of a host gene and regulated by their own promoters. Sometimes miRNAs are transcribed as one long transcript called clusters, which may have similar seed regions, and in which case they are considered a family.The biogenesis of miRNA is classified into canonical and non-canonical pathways

MicroRNA biogenesis and mechanism of action. Canonical miRNA biogenesis begins with the generation of the pri-miRNA transcript. The microprocessor complex, comprised of Drosha and DiGeorge Syndrome Critical Region 8 (DGCR8), cleaves the pri-miRNA to produce the precursor-miRNA (pre-miRNA). The pre-miRNA is exported to the cytoplasm in an Exportin5/RanGTP-dependent manner and processed to produce the mature miRNA duplex. Finally, either the 5p or 3p strands of the mature miRNA duplex is loaded into the Argonaute (AGO) family of proteins to form a miRNA-induced silencing complex (miRISC).

In the non-canonical pathways, small hairpin RNA (shRNA) are initially cleaved by the microprocessor complex and exported to the cytoplasm via Exportin5/RanGTP. They are further processed via AGO2-dependent, but Dicer-independent, cleavage. Mirtrons and 7-methylguanine capped (m7G)-pre-miRNA are dependent on Dicer to complete their cytoplasmic maturation, but they differ in their nucleocytoplasmic shuttling. Mirtrons are exported via Exportin5/RanGTP while m7G-pre-miRNA are exported via Exportin1.

All pathway ultimately lead to a functional miRISC complex. In most cases, miRISC binds to target mRNAs to induce translational inhibition, most likely by interfering with the eIF4F complex. Next, GW182 family proteins bound to Argonaute recruit the poly(A)-deadenylases PAN2/3 and CCR4-NOT. PAN2/3 initiates deadenylation while the CCR4-NOT complex completes the process, leading to removal of the m7G cap on target mRNA by the decapping complex. Decapped mRNA may then undergo 5′−3′ degradation via the exoribonuclease XRN1.

Most studies to date have shown that miRNAs bind to a specific sequence at the 3′ UTR of their target mRNAs to induce translational repression and mRNA deadenylation and decapping. miRNA binding sites have also been detected in other mRNA regions including the 5′ UTR and coding sequence, as well as within promoter regions.The binding of miRNAs to 5′ UTR and coding regions have silencing effects on gene expression, while miRNA interaction with promoter region has been reported to induce transcription. However, more studies are required to fully understand the functional significance of such mode of interaction

The gene regulatory region of TF targets is often complex and can span dozens of kilobases, whereas miRNA-controlled 3′-untranslated regions are, on average, <1 kb in size . Not only does this limit the amount of regulatory inputs that a gene can sample from miRNAs versus TFs, but it also provides less substrate for evolution to “play on,” i.e., to evolve new regulatory inputs, a major driving force in evolution This inherent size restriction may disfavor the evolution of miRNA-dependent gene expression programs. On the other hand, inherent features of miRNA-mediated gene regulation may also provide unique evolutionary opportunities. Whereas, in principle, expression of a gene can become more restricted on an evolutionary time scale by acquiring a novel repressive TF binding site in its promoter, such an acquisition may be constrained by features of the transcriptional activation program