SGH THINK!

What is miRNA?
- miRNA is small non-coding RNA approximately 20 nucleotides in length/, that regulate gene expression post-transcriptionally/ by binding to 3’ UTR, coding sequences or 5’ UTR of target messenger RNA./ It lead to inhibition of translation or mRNA degradation. /MiRNA is estimated that regulate approximately 30% of the human protein-coding genome. /miRNAs control the expression of genes involved in several biologic processes/, including apoptosis, proliferation, differentiation, and metastasis. miRNA came from intron(almost of all miRNA) or exon in DNA. And some protein do their activity then mature miRNAs are formed. Finally they bind to target mRNA then regulate target gene expression. miRNA is found in few years ago.

History of miRNA

- Although miRNA can regulate 30% of human protein genome, scientists didn’t know about miRNA. Before miRNA found, in genetics fields, scientists focused on classic central dogma, which is DNA is transcribed into RNA, which then is translated into protein. This pushed aside the study of all the non-protein-coding sequences. Only in 1993 did the importance of miRNAs begin to be revealed. Lin-4 miRNA was the first miRNA to be discovered in 1993, by the Ambros’s and Ruvkun’s laboratories. They study the nematode C. elegans, heterochronic genes control the temporal development pattern of all larval stages. One of these genes is Lin-4, which is discovered by the isolation of a null mutation that causes a failure in temporal development. Animals with lin-4 loss-of-function mutations are missing some adult structures. Lin-4 activity is required for the transition from the L1 to L2 stage of larval development. In fact, null mutations in Lin-14 gene caused an exactly opposite phenotype of the null-lin-4 mutations. As this figure shows, LIN-14 proteins are expressed in L1 stage. and Lin-4 RNA expression level increase, then LIN-14 protein expression level decrease at stage of L1 to L2 transition. It means, Lin-4 RNA downregulate Lin-14 protein. Upon lin-4 RNA expression, lin-14 protein levels are reduced. Although transcription from the lin-14 gene still occurs, it means lin-4 reaction is posttranscriptional control. Ambros who discovered Lin-4 miRNA found gene which contain lin-4 gene but they couldn’t find the conventional start and stop codons. Then, they concluded lin-4 did not encode a protein. After that, Ruvkun found Lin-14 was downregulated at a post-transcriptional level by lin-4 RNA bind 3’UTR of lin-14 gene. They reported the small and non-protein-coding transcript lin-4 regulates lin-14 protein through its 3’ UTR region. Then a New unexpected cellular regulatory mechanism involving a non-protein-coding transcription had been found.

synthesis of miRNA

- miRNA synthesis consists nuclear processing and cytoplasmic processing. First stage is RNA polymerase reaction for miRNA gene. Almost all miRNA is processed by RNA polymerase II, but small group can be transcribed by RNA polymerase III. The result of RNA polymerase reaction is called primary miRNA simply, pri-miRNA which contain local stem loop structures. After that, pri-miRNA processed by Drosha. Drosha, in conjuction with DGCR8, forms a large complex known as the microprocessor complex. DGCR8 interacts with the ssRNA segment and guides Drosha/ to slice pri-miRNA. Then Dorsha cleaves RNA duplexes about 11bp away from the ssRNA-stem loop junction and thus processes the pri-miRNA to the pre-miRNA with a 5’-phosphate group and an approximately 2 nt at 3’ overhang. Result of Drosha reaction, is called pre-miRNA These are two slicing stages in nucleus. After that, pre-miRNA need to transportation through nuclear pore. The transport of the pre-miRNA occurs through nuclear pore complexes. RanGTP-dependent nuclear transport receptor exporint-5 simply EXP5 that recognizes the over 14-bp double-stranded RNA stem loop with a 3’ overhang followed by cooperative binding to both the pre-miRNA and GTP-bound cofactor Ran in the nucleus. The pre-miRNA bound EXP5 exports out of the nucleus, where hydrolysis of the GTP results in the release of the pre-miRNA. The nuclear cleavage process by Drosha defines one end of the mature miRNA. The pre-miRNA is released in the cytoplasm by EXP5 and is subsequently processed by an endonuclease cytoplasmic Rnase III enzyme Dicer/ to create a mature miRNA. Dicer works in close proximity with other proteins. In human, Dicer is associated with two closely related proteins, TRBP full name is trans-activation response RNA-binding protein, and protein kinase PACT. At Dicer cleavage, the miRNA duplex is incorporated into an Ago family protein complex. This generates an effector complex. Dicer, TRBP, PACT and Ago protein contribute to RNA-Induced Silencing Complex assembly by forming a RISC loading complex. The exact mechanism regarding to the role of RISC Loading Complex in RNA loading to Ago is not known. However, the stable end of the miRNA duplex binds to interacting proteins in the RLC, and the unstable end associates with the Ago proteins. Then Ago protein remove the miRNA passenger strands. In figure miRNA star. After loading, the miRNA guides the RISC to its target mRNA.

mechanism of miRNA

- There are two methods of gene expression regulation of miRNA which are translational repression, and mRNA degradation. The degree of miRNA-mRNA complementarity is a major determinant of the regulatory mechanism process. if miRNA and mRNA bind with perfect pairing, then mRNA degradation process occurs. other case, if miRNA and mRNA bind partially, then mRNA translation be blocked by miRNA complex. First mechanism is mRNA degradation. This caused by perfect base-pairing between target mRNA and miRNA. High degree of complementarity facilitate Ago protein’s slicer activity. AGO protein interact with a GW182 protein, which in turn interacts with cytoplasmic poly-(A)-binding protein(PABPC) and the cytoplasmic deadenylase complexes PAN2-PAN3 and CCR4-NOT. GW182 protein consist of an aminoterminal AGO-binding domain(ABD), and a silencing domain(SD). The PANS2-PAN3 and CCR4-NOT complexes catalyze the deadenylation of the mRNA target. Then 3’ to 5’ degradation process by the exosome occurs. Or deadenylated mRNAs are decapped by Dcp1 and 2 decapping enzymes then rapidly degradaded by 5’ to 3’ exoribonuclease (XRN1). mRNA degraded by RNA interference pathway then translation can not be occurred, finally target gene expression be downregulated. Second is Translational repression, which is caused by low degree of complementarity. For translation, need RNA processing which are 3’ end poly A tailing, 5’ capping, circularization, ribosome binding and so on. Mature miRNA can block some of these processes. There four models to inhibit translation by miRNA. First model is miRNA complex bind to target mRNAs and repress initiation at the cap recognition stage. miRNA complex compete for eIF4 which recognize mRNA 5’ terminal cap, then failure of the translation initiation process. Second model is miRNA complex bind to target mRNAs at the 60S ribosomal recruitment stage. After 40S ribosomal subunit assembly at the mRNA to enable the preinitiation complex by eIF3 and eIF4G, the elongation process is initiated by joining of the 60S ribosomal subunit at the AUG codon of the mRNA and the 40S preinitiation complex. but miRNA complex inhibit the assembly of the 60S ribosomal subunit, then translation cannot be started. Third model is miRNA complex can prevent mRNA circularization. In eukaryotic translation, the mRNA molecule becomes circular for improve translation efficiency by eIF4G and eIF3 interaction with poly A binding protein. miRNA complex block mRNA circularization then translation efficiency decrease. The last model is miRNA complex attachment to target mRNAs facilitates premature separation from ribosomes. The accumulation of mRNA targets of miRNAs and the Ago proteins in P-bodies argues that miRNAs are increasing the amount of ribosome-free mRNA, then target mRNA translations are repressed.