Lecture 17 – miRNAs in Plants & Animals BIOL 5190/6190 Cellular & Molecular Singal Transduction Prepared by Bob Locy Last modified -13F

Introductory miRNA synthesis & function Plant paradigm Animal paradigm siRNA synthesis & function

Module 11: Cell Proliferation Figure 1. Major biogenesis pathways of small RNAs from inverted repeat transcripts in plants and animals. (a) In plants, canonical microRNAs (miRNAs) are produced by the nuclear RNase III Dicer-like1 (DCL1), which cuts from the base of the hairpin towards the loop; a subset of plant miRNAs are processed from the loop towards the hairpin. One miRNA/miRNA* duplex is shown, but there can be several such duplexes depending on the length of the stem. These are transported from the nucleus via HASTY, an Exportin5 (Exp5) homolog, for loading into an Argonaute (AGO) complex. The main miRNA effector in plants is AGO1, and to a lesser extent AGO10 and other AGOs; AGO7 carries the exceptional miRNA miR390. Long well-paired hairpins (proto-miR/inverted repeat (IR) transcripts) can be processed by a diversity of Dicers to generate either miRNAs or small interfering RNAs (siRNAs). The subcellular location for dicing by DCL2 and DCL4, and subsequent AGO loading of the resulting siRNAs, is not yet clear. nt, nucleotide. (b) In animals, canonical miRNAs are processed by the nuclear RNase III enzyme Drosha. The precursor miRNA (pre-miRNA) hairpin is exported to the cytoplasm by Exp5 to generate a single miRNA/miRNA* duplex, which is loaded into a miRNA class AGO protein (Drosophila dAGO1, Caenorhabditis elegans ALG1/2, or vertebrate Ago1 to Ago4). There are Drosha-independent non-canonical pathways, including the mirtron pathway where intron splicing and lariat debranching generate pre-miRNA hairpins. Also, vertebrate miR-451 is matured by a Dicer-independent route. Here, Drosha cleavage generates a short hairpin that is loaded into the 'Slicer' Ago2, which cleaves its 3' arm; this is resected to yield the mature miRNA. Unlike other vertebrate miRNAs, miR-451 can only be matured in Ago2. Finally, in the Drosophila hairpin RNA pathway, long inverted repeats are processed by the endogenous siRNA pathway, being cleaved by d-Dicer2 to generate siRNAs that load dAGO2. Many Drosophila miRNA* species are also preferentially sorted into dAGO2 (dashed arrow).

Module 04: Animal miRNA biogenesis Biogenesis and function of microRNA.
RNA polymerase II (Pol II) transcribes miRNA genes to form primary miRNA (pri-miRNA) transcripts, which has a characteristic stem-cell structure. The latter is recognized by the ribonuclease Drosha that acts together with DiGeorge syndrome critical region 8 (DGCR8, also known as Pasha) to cleave pri-miRNA to form pre-miRNA, which is then exported from the nucleus by Exportin 5. Once it enters the cytoplasm, the pre-miRNA is recognized by TAR RNA-binding protein (TRBP) and the ribonuclease called Dicer, which cleaves the precursor to form the mature microRNA (miR). The miR acts by binding to Argonaute (Ago1–4) proteins to form a RNA-silencing complex (RISC) that recognizes and inhibits target messenger RNAs (mRNAs) through a number of mechanisms, such as translational repression, mRNA deadenylation and mRNA degradation. The first step in microRNA biogenesis is the transcription of the miR gene by RNA polymerase II (Pol II) to form the primary miRNA (pri-miRNA) transcripts, which has a characteristic stem-loop structure and a 5′ capped polyadenylated (poly A) tail (Module 4: Figure microRNA biogenesis ). This pri-miRNA is recognized by Drosha, which is a double-stranded RNA-specific nuclease that acts together with DiGeorge syndrome critical region 8 (DGCR8, also known as Pasha) to cleave pri-miRNA to form pre-miRNA. This pre-miRNA is then exported from the nucleus by Exportin 5, which is a RAN-GTP-dependent nuclear transport receptor. Once it enters the cytoplasm, the pre-miRNA is recognized by the transactivator RNA-binding protein (TRBP) and the ribonuclease Dicer, which cleaves the precursor to form the mature microRNA (miR).The miR acts by binding to Argonaute (Ago1–4) proteins to form a RNA-silencing complex (RISC) that recognizes and inhibits target messenger RNAs (mRNAs). A short 'seed region‘ between bases 2 and 7 at the 5′ end of the miRNA is responsible for recognizing and binding to the 3′ untranslated region (UTR) of their target mRNAs to inhibit protein synthesis through a number of mechanisms such as translational repression, mRNA deadenylation and mRNA degradation. The translational repression of protein synthesis can occur through inhibition of either EIF4E to prevent initiation or the ribosomes to block elongation. Expression can also be inhibited by deadenylation of the poly-A tail by activation of the CCR4-NOT. The RISC complex can also bring about direct degradation of mRNA. Cell Signalling Biology - Michael J. Berridge - www.cellsignallingbiology.org - 2012

Animal miRNA Function - Overview The task of establishing microRNA properties and function of individual miRs is ongoing and already there are indications that each miR can modulate the activity of up to 100 mRNAs to influence a large number of key biological processes: Maintenance of embryonic stem (ES) cell pluripotency. MicroRNA modulation of cell-cycle regulatory mechanisms p53 functions and microRNAs MicroRNA regulation of differentiation Differentiation of cardiac cells Differentiation of smooth muscle cells Cell proliferation Apoptosis Stress responses. MicroRNA dysregulation and cancer

Module 04: Animal miRNA biogenesis Embryonic stem (ES) cell miRNAs.
The survival and self-renewal of embryonic stem (ES) cells is maintained by a limited number of pluripotency regulatory transcription factors such as Oct4, Sox2, Nanog and Krüppel-like factor 4 (Klf4) (green panel). These regulatory factors use a number of ES cell microRNAs (orange panel) to control the expression of key proteins responsible for the cell-cycle regulatory mechanisms (pink panel). The blue panel at the bottom illustrates some of the microRNA regulatory processes that control differentiation. Cell Signalling Biology - Michael J. Berridge - www.cellsignallingbiology.org - 2012

Module 04: miRNAs and p53 MicroRNAs and p53 function.
There are intimate relationships between the functions of microRNAs (miRs) and p53. The expression of p53 is regulated by miR-125b and miR-380-5p, whereas activated p53 can control the transcription of a number of miRs that contribute to regulation of cell cycle arrest and apoptosis. Cell Signalling Biology - Michael J. Berridge - www.cellsignallingbiology.org - 2012

Module 08: Cardiac Development Development of cardiac cells.
The myogenic differentiation transcription factors serum-response factor (SRF), myocyte-enhancer factor 2 (MEF2) and MyoD activate the expression of the cardiac genes that emerge during the process of myoblast differentiation. These transcription factors also activate the miR-1 and miR-133 clusters and these two miRNAs contribute to the process of differentiation by helping to suppress cardiomyocyte proliferation and to fine-tune the expression of many cardiac components such as those functioning in cardiac conduction. Differentiation of cardiac cells Cardiac cells are some of the first functional cells to appear during development. A limited number of myogenic differentiation transcription factors such as serum-response factor (SRF) and myocyte enhancer factor 2 (MEF2) are responsible for activating the expression of the cardiac genes that emerge during the process of myoblast differentiation (Module 8: Figure cardiac development ). In addition to activating these cardiac-specific genes, these transcription factors also activate the bicistronic miR-1 and miR-133 clusters, and these two miRNAs contribute to the process of differentiation by helping to suppress cardiomyocyte proliferation and to fine-tune the expression of many cardiac components such as those functioning in cardiac conduction. There are multiple loci responsible for encoding miR-1 (miR-1-1 and miR-1-2) and miR-133 (miR-133a-1, miR-133a-2 and miR-133b). The genes located at these different loci are highly related and genetically redundant and will thus be referred to as miR-1 and miR-133 for simplicity. The organization of the miR-1-1/miR-133a-2 cluster is illustrated in Module 8: Figure cardiac development . The myogenic transcription factors act at both upstream and intronic enhancers to increase the expression of these two miRNAs that contribute to the emergence of the cardiac phenotype. One of the actions of miR-1 is to operate in a feed-forward regulatory loop to inhibit histone deacetylase 4 (HDAC4), which is a transcriptional repressor of MEF2.Both miR-1 and miR-133 help to reduce cardiomyocyte proliferation, with miR-1 acting to inhibit translation of the heart and neural crest derivative-2 (Hand2) protein that promotes proliferation of the early myocyte population. The miR-133 also inhibits proliferation by reducing the expression of cyclin D, which has a critical role in activating the cell cycle (Module 9: Figure proliferation signalling network).Cardiac microRNAs also play a role in regulating development of the conduction system, which consists of numerous ion channels. For example, miR-1 regulates the gap junction protein connexin 43 (Module 8: Figure cardiac development ), which provides the ionic communication necessary for the action potential to spread throughout the heart to drive each contraction. Expression of Kir2.1, which provides the IK1 current that holds the cardiac resting potential in its hyperpolarized state (Module 7: Figure cardiac action potential), and the HCN2 and HCN4 members of the hyperpolarizing-activated cyclic nucleotide-gated (HCN) channel family, is also regulated by miR-1. The miR-133 also plays a role in the development of the conduction system by modulating the expression of the HCN2 and the Kv11.1 channel [also known as the human ether-a-go-go (hERG) channel]. Cell Signalling Biology - Michael J. Berridge - www.cellsignallingbiology.org - 2012

Module 08: Smooth Muscle Cell Differentiation Control of smooth muscle differentiation.
Smooth muscle cells (SMCs) are highly plastic in that they can readily switch from a highly differentiated contractile smooth muscle phenotype to a more fibroblast-like proliferative phenotype. Serum-response factor (SRF), working together with the miR-143/miR-145 cluster, controls this proliferation/differentiation switch. Differentiation of smooth muscle Smooth muscle cells (SMCs) are unique among the muscle cells in that they are able to switch rapidly between differentiation and proliferation. Smooth muscle cell proliferation plays an important role in wound healing, but also occurs during vascular remodelling, which can have serious pathophysiological consequences when it leads to pulmonary vasoconstriction and hypertension (Module 9: Figure SMC proliferation). Proliferation is activated by Ca2+ and by the MAP kinase signalling pathways (Module 8: Figure smooth muscle cell differentiation ). In the latter case, MAP kinase acts through ERK1/2 to phosphorylate the E twenty-six (ETS) transcription factor Elk-1 (Module 4: Figure ETS), which then begins to activate the genes responsible for proliferation. The Krüppel-like factor 4 (Klf4) also plays a role in stimulating proliferation and it also acts to inhibit myocardin, which is a major SMC differentiation factor.A key component of the proliferation/differentiation switch is an increase in the level of serum-response factor (SRF) that binds to the CarG box, where it recruits the coactivator myocardin and this complex is responsible for the expression of SMC genes. The switch to differentiation coincides with a cessation of proliferation and this process is orchestrated by SRF-induced activation of the miR-143/miR-145 cluster, which act to inhibit many of the components that promote proliferation (Module 8: Figure smooth muscle cell differentiation ). The miR-143 inhibits the stimulatory action of Elk-1, whereas miR-145 enhances the activity of the differentiation factor myocardin while simultaneously removing the inhibitory action of Klf4. Inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) also reduces the Ca2+-dependent activation of proliferation. Cell Signalling Biology - Michael J. Berridge - www.cellsignallingbiology.org - 2012

miRNAs in Cancer

miRNAs in Cancer The roles of p53-regulated miR-200c in EMT and stem-cell-like properties. p53 directly binds to the miR-200c promoter and activates its expression. The elevated miR-200c hinders EMT via ZEB1 and reduces cell populations with stem-cell-like properties by BMI1. These pathways prevent the formation of metastatic cancer cells. Chen et al. Journal of Biomedical Science 2012 19:90   doi:10.1186/1423-0127-19-90

miRNAs & Cancer Canonical biogenesis pathway and mechanisms of miRNA deregulation. After RNA polymerse II-dependent transcription, pre-miRNAs are generated from pri-miRNAs or spliced RNA by Drosha-DGCR8 complex or intron splicing pathway, respectively. After exporting to cytoplasm, Dicer digests the pre-miRNAs to the mature miRNAs which guide the miRISC to inhibit target mRNAs. Factors in the yellow box indicate protein regulators or mechanisms leading to the aberrant biogenesis of miRNAs in cancer. See text for more detailed description.

The role of plant hormones in regulating the interaction between biotic and abiotic stress. The role of plant hormones in regulating the interaction between biotic and abiotic stress. The schematic diagram shows cross-talk occurring between hormones, transcription factors, and other regulatory components when biotic and abiotic stresses occur concurrently. This complex network of interactions allows plants to respond in a highly specific fashion to the exact combination of environmental stresses encountered. Grey arrows show induction or positive regulation, while orange bars show inhibition or repression. Events characteristic of abiotic stress responses are shown in pink, while those characteristic of biotic stress responses are shown in green. Transcription factors and other regulatory genes are represented by orange boxes. ROS, reactive oxygen species; ABA, abscisic acid; JA, jasmonic acid; SA, salicylic acid; PR, pathogenesis-related; SAR, systemic acquired resistance; HSF, heat shock factor. Atkinson N J , and Urwin P E J. Exp. Bot. 2012;jxb.ers100 © The Author [2012]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Embryonic stem (ES) cell miRNAs Embryonic stem (ES) cell miRNAs.
The survival and self-renewal of embryonic stem (ES) cells is maintained by a limited number of pluripotency regulatory transcription factors such as Oct4, Sox2, Nanog and Krüppel-like factor 4 (Klf4) (green panel). These regulatory factors use a number of ES cell microRNAs (orange panel) to control the expression of key proteins responsible for the cell-cycle regulatory mechanisms (pink panel). The blue panel at the bottom illustrates some of the microRNA regulatory processes that control differentiation.