Micro RNA

By- Sai Kriti Arora MSc ( 2nd sem)

WHAT IS miRNA?
Class of non coding regulatory RNAs Have 21-23 nucleotides sequence Bind to target RNA and leads to its silencing MicroRNAs were first discovered in 1993 by Victor Ambros, Rosalind Lee and Rhonda Feinbaum Study was related to the development in the nematode Caenorhabditis elegans (C. elegans) regarding the gene lin-14.

This screen led to the discovery that the lin-14 was able to be regulated by a short RNA product from lin-4 Lin-4 gene transcribed a 61 nucleotide precursor that matured to a 22 nucleotide mature RNA which contained sequences partially complementary to multiple sequences to the 3 untranslated regions of the lin-14 mRNA. This complementarity inhibited the translation of lin-14 mRNA

miRNA NOMENCLATURE
Names are designated to specific miRNAs before publication of their discovery Experimentally confirmed microRNAs are given a number that is attached to the prefix mir, for eg: mir-123 The uncapitalised mir- refers to the premiRNA and the capitalised miR- refers to the mature form. miRNAs with similar structures at 1 or 2 nucleotides are annotated to show their similar structure with added lower case letter eg miR-1a and miR-1b.

It must also be preceded by the annotation for the species they are observed in eg: for Homo sapiens, it is hsa-miR-xxx. Other common miRNA species include viral v-miRNA and Drosphila d-miRNA. MicroRNAs originating for the 3 end or 5 end are denoted with a -3p or 5p suffix eg. miR-142-5p, miR-142-3p

SYNTHESIS OF miRNA
miRNAs are generated by two RNA cleavage reactions from a longer RNA transcript known as pri-miRNA Pri-miRNA has a hairpin like structure These two cleavage reactions require two disinct RNases- DICER and DROSHA During first cleavage, stem loop is liberated known as pre-miRNA ( by Drosha) Second cleavage generates mature

Characteristic of these enzymes is that they recognize RNAs on the basis of their structure rather than their specific sequence Drosha is the member of RNase III family Makes two cleavages that cut pre-miRNA from pri-miRNA Works together with an essential specificity sub-unit protein known as Pasha or DGCR8 (Di Georges syndrome critical region gene 8) in some organisms Drosha and Pasha constitute an active microprocessor complex

The base paired stem in the pri-miRNA is about 33 bp and has a terminal loop of variable size at the top Stem region can be divided into two functional segments: 11 bp lower stem and 22 bp upper stem REQUIREMENT FOR DROSHA ssRNA is needed flanking 5 and 3 end of stem loop these ssRNA and dsRNA junctions are responsible for determining the cleavage specificity of Drosha Drosha cleaves 11 bp away from ssRNAdsRNA junctions i.e between upper and lower

Drosha resides in cells nucleus The pre-miRNA generated by Drosha in nucleus is 65-70 nucleotides long containing of dsRNA and the top loop Drosha cleaves dsRNA in a manner that leaves a 2 nucleotide overhang on the 3 ends of the dsRNA product This 3 overhang is important for recognition of the RNA molecule by next enzyme i.e. DICER The pre-miRNA liberated by Drosha is exported to cytoplasm where Dicer carries out second cleavage

DICER

STRUCTURE OF DICER
Consists of three modules: two Rnase III domains and a ds RNA binding domain called PAZ domain(Piwi, Argonaute, Zwille) PAZ domain is at the bottom of the handle forming a binding pocket for the 3 end of ds RNA PAZ domain anchors the 3 terminus of RNA to position the active sites of the enzyme 22 nucleotides away in a ruler like fashion

Each RNase domain carries an active site and is responsible for cleaving one of the two strands of RNA Dicer can act on any ds RNA regardless of sequence and cleaves RNA 22 nucleotides from its end

MECHANISM OF REGULATION OF GENE EXPRESSION

Expression of genes is inhibited in three ways: a. Destruction of target mRNA b. Inhibition of translation c. Chromatin modifications that inhibit transcription

The active form of miRNA is the single stranded form(guide strand) incorporated into a RISC( RNA induced silencing complex) protein complex The miRNA must be denatured to give a guide RNA and a passenger RNA Guide RNA gives specificity to RISC and passenger strand usually gets discarded The guide RNA base pairs with the target mRNA and thus promotes silencing of gene expression The base pairing between miRNA and target RNA is initiated by seed residues which are sequences between base 2 and 9 of the miRNA

This mature RISC is targeted to target RNA containing sequences complementary to guide RNA The main component of this regulatory complex is Argonaute protein which mostly is RNA cleaving enzyme There are 8 Argonautes in humans but not all of these have slicer activity Argonaute has a PAZ domain and an Rnase domain PAZ domain recognizes 3 end of the guide RNA which is base paired to the target RNA This binding positions the active site of the RNase domain to cleave the target RNA strand

If sequences are highly complementary, then the target is degraded (slicing) If there are mis-matches in sequence, then there is mostly inhibition of translation RISC can also be directed to the nucleus where it recruits other proteins that modify the chromatin around the promoter of the gene This leads to silencing of transcription in which centromeres are transcribed to produce RNAs that hybridize with other RNAs from the same region These dsRNAs are acted upon by Dicer

Mechanism of translational repression is still under scrutiny but it appears that miRNAs lead to the sequestration of mRNA in processing bodies(P bodies) in cytoplasm where translation is repressed Binding of miRNA can also destabilize the poly-A tail of target mRNA disrupting translation

DIFFERENCES

miRNA It is endogenously present in cells May be slightly shorter It is single stranded Binds to non-coding part of mRNA and acts as a signal to prevent translation

siRNA It can be synthesised outside the cells Has 20-25 nucleotides It is formed from two complementary strands Attaches to a coding region of mRNA and physically blocks translation

APPLICATIONS OF miRNAs
Have a central role in gene regulation Can be used as biomarkers A biomarker is a physical sign or cellular, bio chemical, molecular or genetic alteration by which a normal or abnormal biologic process can be recognized or monitored, or both, and that might have diagnostic or prognostic utility miRNA posseses features of an ideal biomarker: they reflect the physiological or pathological state of cells and tissues, are relatively stable,and can be detected by various methods

miRNAs have been implicated in development, physiological functions and disease processes Skin morphogenesis, pancreas development, muscle differentiation, cardiac growth and neural development are all processes in which miRNAs have been implicated. A large number of malignancies have been associated with altered miRNA expressions. other nonmalignant diseases in which miRNAs have been implicated include: alzheimers disease neuro psychiatric disorders viral infections such as hepatitis AIDS primary biliary cirrhosis systemic lupus erythematosus rheumatoid arthritis

Aberrant expression of miRNAs can be caused by chromosomal abnormalities, insertion of foreign genetic material such as viral genomes, mutations or single nucleotide polymorphisms (SNPs) Another characteristic of miRNAs is the presence of mutations and polymorphisms, not only in the sequences of the mature miRNAs,but also in the sequences of the pri-miRNAs and the pre-miRNAs These sequence changes can affect the biogenesis of the miRNAs or the action of mature miRNAs on target sequences. It is important for the use of miRNAs as biomarkers, since one should be able to discriminate between those miRNAs with polymorphisms and those without to allow the precise measurements of the levels of the mature miRNAs.

Advantages of miRNAs as biomarkers over mRNA or proteins

A single mRNA is usually translated into a single protein, but a single miRNA is capable of regulating the translation of many genes So as compared with mRNA expression analysis, a limited number of miRNAs can be used as biomarkers, thereby reducing the complexity miRNAs appear to be long-lived in vivo, are stable and relatively resistant to

miRNA and cancer

Cancer cells and tissues have different miRNA profiles than normal cells and tissues, suggesting that miRNA profiles could be used for the diagnosis of cancer In addition to diagnosis, miRNA analysis has also been tested as a tool for the early detection of cancer or cancer recurrence, cancer classification In colon adenocarcinomas; high levels of mir21 were associated with a poor therapeutic outcome and with poor survival

Hsa-miR-205 is used as a biomarker in squamous cell lung carcinoma Elevated levels of various serum miRNAs have been detected in prostate,ovarian and lung cancers in comparison with normal serum Circulating exosomal miRNAs can also be used as biomarkers in cancer. Exosomes are small membrane bound vesicles released by cells, which are an important source of cell free miRNA in serum and other bodily fluids, such as

miRNA and autoimmunity

miRNAs in normal immune functions

miRNAs in autoimmune disorders

miRNAs in heart diseases

Profiling of miRNA expression in normal tissues has revealed that a subset of miRNAs are expressed selectively in striated muscles miR-1, miR-133, miR-499, and miR-208a were significantly increased in plasma as soon as 1 hour after myocardial infarction, suggesting that these miRNAs may be useful in the diagnosis of acute myocardial infarction Upregulation of miR-21 in diseased cardiac fibroblasts has been shown to promote cardiac fibrosis

miRNA as therapeutic tools

miRNA therapies could involve administration of a specific miRNA to downregulate specific target genes or the blocking of certain miRNA to increase expression of target genes antimiRs and miRNA mimics can be used as therapeutics antimiRs are antisense oligonucleotides with reverse complementary sequence of the target miRNA These bind to mature miRNA and inhibit its activity These have proven useful for the inhibition of miR122 in liver

miRNA mimics are synthetic RNA duplexes in which one strand is identical to the mature miRNA guide strand Difficulties in using miRNAs as therapeutic agents: May have some off-target effects Systemic delivery of antimiRs and miRNA mimics may cause secondary effects like altered blood pressure or change in levels of circulating hormones