Non-coding RNAs and Gene Regulation

• mRNAs perform functions as RNA molecules, not coding for proteins
• microRNAs regulate gene expression
• short RNAs (siRNA) derive from processing longer precursor mRNAs
• Long non-coding RNAs (IncRNA) exist
• Circular RNAs are a type of non-coding RNA
• Piwi-associated RNAs (piRNAs) are another type of non-coding RNA
• Non-coding RNAs can act as signaling molecules
• Transfer RNA is a non-coding RNA

Small RNAs in Gene Regulation

• microRNA-mediated gene regulation is a key process
• RNA interference (RNAi) with chromatin also regulates genes
• PIWI proteins play a role in gene regulation

microRNA biogenesis stages

• Primary microRNA (pri-miRNA) is transcribed from an mRNA gene
• Drosha, an endonuclease, processes pri-miRNA in the nucleus to form precursor microRNA
• Pre-miRNA is transported from nucleus to cytoplasm through Exportin 5
• Dicer cleaves pre-miRNA into microRNA duplexes
• Ago endonuclease loads one strand of the microRNA duplex into the RISC complex
• Mature miRNA guides RISC (RNA-induced silencing complex) to complementary sequences on target mRNA
• Gene silencing achieved through translation inhibition and mRNA degradation

Small RNA regulation

• The Dicer-associated complex processes double-stranded RNAs into small RNAs like in vitro synthesized dsRNA, miRNA precursors, transgene dsRNA, viral dsRNA, transposon dsRNA, and heterochromatic dsRNA
• Argonaute-associated complexes (RISC and RITS) mediate mRNA degradation, translation inhibition, mRNA cleavage, and heterochromatic domain formation

Features - miRNA vs siRNA vs piRNA

• miRNA length ~22 nucleotides
• siRNA length ~21 nucleotides
• piRNA length 24-30 nucleotides
• miRNA uses Drosha or Dicer to process
• siRNA uses Dicer to process
• piRNA uses Zucchini & unknown trimming enzymes
• miRNA, siRNA, & piRNA all use AGO
• piRNA uses PIWI
• miRNA acts by translational repression and mRNA degradation
• siRNA acts by cleavage via RNA
• piRNA acts by transcriptional/post-transcriptional repression of transposons and multigenerational epigenetic phenomena
• miRNA regulates protein-coding genes
• siRNA regulates coding genes and transposons, and has antiviral defense properties
• piRNA regulates pre-pachytene piRNA which is transposon silencing, pachytene piRNA which is unknown, and small RNA in soma

Discovery of MicroRNAs in C. Elegans

• Heterochronic gene lin-4 encodes a small RNA antisense to lin-14 and found lin-4 does not encode protein
• small RNA was antisense complementary to regions of lin-14 mRNA.
• Post transcriptional regulation happens in heterochronic gene lin-14. lin-4 mediates temporal pattern formation
• Non-coding genes are essential for development
• lin-4 knockout causes failed vulva development (larvae cannot lay eggs)
• lin-14 mutations cause developmental defects in adults

Control of C. elegans development timing

• Genes related mapped to developmental processes that were non-coding
• Knockout Lin 4 = eggs laid with a failure for vulva development
• lin-14 = had developmental in adults
• lin4 and 14 linked

Lin-4 and Lin-14 Complementarity

• lin4 overexpression reveals smaller RNA fragments (precursor of mRNA)
• lin4 silences lin14 by sequence complementarity between non-coding RNA and the free prime untranslated region

Broader RNAi Phenomenon

• The "anti-coordinator" effect increases mRNA levels but decreases protein expression due to microRNA-mediated repression, adding complexity to gene regulation
• Introducing lin-4 (miR-125) decreases lin-14 expression, demonstrating microRNAs block gene translation, reducing protein levels
• This is RNA interference (RNAi)- small RNAs controlling gene expressions
• miRNA can simply block translation

microRNA Transcription

• Typically transcribed by RNA Polymerase II
• Long (>1 kb) has highly structured primary microRNA (pri-miRNA) transcripts; can also have dual role as an mRNA
• Introns can be coding or non-coding transcripts, exonic regions too
• miRNA loci are often polycistronic (e.g., miR-17-92 cluster)
• Some viral miRNAs transcribed by RNA Polymerase III

Microprocessor Complex

• Made of Drosha + Dicer
• Drosha (RNase III-type endonuclease) processes pri-miRNA into ~65 nt stem-loop precursor miRNAs (pre-miRNA) with 5' phosphate and 2nt, 3' overhang
• DGCR8 as essential cofactor
• Pre-miRNAs are exported to cytoplasm by Exportin 5

Cytoplasmic pre-miRNA processing

• Dicer (RNase III-type endonuclease) with 2 active cutting sites processes pre-miRNA into small RNA duplexes with 5' phosphate and 2nt, 3' overhang
• TRBP cofactor, processing eff & tunes length
• Preference for Drosha products (2nt, 3' overhang)
• PACT acts as a cofactor, role unclear

The Argonautes

• Multi-domain regulate gene expression
• structure = "if it looks like a duck & it quacks like a duck, it must be a duck"
• hAgo2 mediates microRNA interactions (slicing activity)
• MID (binds to 5' end, U/A) needs Mg2+ for stability
• PIWI backbone, miRNA seed: A-form helix (cuts RNA)
• PAZ anchors 3'end of miRNA

Argonaute Cleft

• Holds the target mRNA for regulation
• RNA-induced silencing complex (RISC) assembly:
• Loading (ATP) loads miRNA duplex into Ago protein [double-stranded] Unwinding occurs (NO ATP “rubber band” model)
• Mature guide miRNA vs passenger strand [One strand (passenger strand) is removed]
• Stability at 5' end with preference for 55' U dependent
• Mismatches* in miRNA duplex @ 2-8 ["seed region"] & 12-15 promote unwinding

Asymmetry Rule

• The strand with the less stable 5' end (usually with a U base) chosen as the guide strand as its easier for uniwnd & load to AGO, b) MID domain preference to U/A & C) seed region to form base pairs easily

Summary - Argonaute Functions

• Binds to single strands of either 21nt siRNAs or miRNAs
• 5'phosphate bound by ID
• 5' most nts MID & PIWI
• 3'end bound by PAZ
• PIWI fold into an RNaseH domain & carries out “slicer" activity (axe)
• RISC work with 'scan' RNAse in cells for complementarily
• Extensive base-pairing w target, base- pairing at nt 10-11 triggers a conformational change & slicing.

Targeting Using Base-Pairing

• miRNAs using Target mRNAs other parameters for miRNA targeting:
• 3' UTR that can enhance or prevent miRNA binding
• Distance from ORF [open reading frame] & polyA tail effecting positioning
• RBP sites in 3' UTR. Some RBPs compete with or assist miRNA binding
• miRNAs can cooperate by the same 3' UTR being bound multiple miRNAs which enhances repression

Non-Canonical Pathways

• MiRNA follows a strict processing route from transcription to Argonaute loading as a conical, however in a non-canonical way, RNAs don't follow biogenesis but still regulate gene expression via Argonaute

GW182 Proteins

• Act as either silencing effector or facilitator
• Interact directly & downstream of AGOs.
• Direct tethering of GW182 to mRNA leads to translation suppression & mRNA destabilization

GW182 Functions

• Strong interactor & scaffolding protein & block ribosome binding to the mRNA at the cap or start site
• If translation already begun, GW182 interacts with ribosomes, preventing proper elongation/ premature translation termination = mRNA destabilization & degradation
• The miRNA-AGO-GW182 complex: silences genes by blocking translation or causing mRNA decay
• miRNAs + AGO + GW182 = mRNA silencing & gene regulation

Methods to Detect mRNA Stability

• 5'capped radiolabeled target mRNA + miRISC + ribosome subunits test
• IF RNA ONLY blocks translation = mRNA still there but NO protein is made, so it is not destabilizing mRNA. Only preventing the transfer of mRNA = use western plot (look at proteins)
• IF RNA disappears = miRNA causes it to disappear from a destabilizing action AND mRNA is silencing translation by destabilizing the mRNA =northern blot or quantitive PCR can be preformed.
• Example: As Let-7 levels increase, Lin41 mRNA levels drop bc Let-7 miRNA targets Lin41 mRNA- becomes degraded from destabilzation

MicroRNA-mediated deodenyaltion and RNA destabilizaiton

• MicroRNA recruits GW182
• GW-182 brings CAF1, CCR4 and NOT1 which cut of polyA tail = Deadenyaltion
• GW182 recruit DCP1/2 enzymes & removes 5' cap= decapping = degradation = once cap & tail gone the RNA is destroyed by nucleases (Xrn1 & exosomes)

Role of GW Complexes

• GW182 brings a complex of CAF1, CCR4 and NOT1 & destabilization happen
• After miRNA binds target mRNA through AGO, GW182 recruited to mRNA and more degradation process
• Process bodies create a place where microRNA functions by storing and sequestering it away from ribosomes, GW and Dcp bodies are membranous organelles.

Processing bodies

• Areas the are gel like and promote mRNA turnover through GW182, CCR4, NOT, DCP1, Xrn1 [enyzmes that break donw mRNA] and deactivate deadenylaitons can then decap mRNA

Endogenous RNAi

• Centred miRAN sites act a newer way for miRNAs to target mRNAs:
• miRNA targeting code has a functional site that's centred paired allowing “Silciing"

Summary Points About microRNA Interaction

• limited miRNA = mRNA base-pairing prevents 'sicling'
• miRISC triggers rapid deadenylation of target mRNA
• mRNA can then be de-capped by DCP1/2 enzymes, which are activated by their cofactors, though the -GW182 & CCR4/ NOT deadenylation complex [CAF1]
• mRNA is turned over by scavenging with nucleases [exosome & Xrn1/2] & are regulated by mRNAs to promote growth, differentiation and in cancer

MicroRNA Biogenesis

• Small, non-coding RNAs bind mRNA + prevent proetin production to control gene expression, happening both nuclear and cytoplasmicly:
• miRNA, which is transcribed by RNA polymerase II, which turn into pri-miRNA that is the cuts pre-miRNA that then moves into cytoplasm
• Dicer cuts pre-miRNA, binds target mRNA from binding and triggering mRNA degradation that block translation.
• microRNA follows a strict processing route from transcription to Argonaute loading follows as a conical but if not this then = RNAs don't follow biogenesis but still regulate gene expression via Argonaute

microRNA Regulaiton

• single miRNA: regulates its expression, binds to specific mRNA by the process, leading to mRNA degradation or inhibition [miR-21]
• Single miRNA targets to multiple mRNA; multiple mRNA is affected simultaneously and since miRNA recognize short seed sequence in 3' UTR multiple are regulated
• Target multiple immune related genes [inflamation &cancer]

Functions for Regulaiton

• Target muliple nodes within proteins or kinases by NFB = miRNA which target kinase involved in phosphorylation while targeting proteins within same pathway
• miRNAs can influence through transitional network; miRNAs is modulated with TF which regulate multiple genes due its high # of targets but creates a feedback loop

Signalling

• Participate within transcriptional activator when cell responds by signal accordingly to it's program
• Signals activate tranicitpon/ cofactor of gene 1, howver program cnat be expressed at cell since miR-A repressed and can only be on in OFF mode

Model of Transcriptional Default Repression

• Repressor (R) inhibits the activity of the pathway activator in the absence of the signal
• Signaling relieves this inhibition, activating transcription.
• miRNAs contribute to default repression by minimizing mRNA production

Role of miR-125b in DNA Damage

=example of microRNA as mediator of default responses

• Normal cell (left panel), miR-125b targets, apoptosis activity
• Genotoxic (right panel), p53 activated, induces apoptosis
• p53 = helps apoptotic DNA damage
• miR-125b= reduced, preventing deaths damage if not fit in mutations or the opposite and can self destruct apoptoticly, but
• 125b = default repressor switiching Off on DNA damage

High Signal Act of Hippo Signal

• Hippo Off= target genes become active
• Middle panel, high= LATS phosphylates the prevents to prevent with with 3723s
• miR372 &373 kinase LAT= tranc ON

MicroRNAs and Yap/TAz

• regulators of cell growth, proliferation controlled by Hippo signaling pathway (tumor suppressor)
• When hipoo OFF = enters enter & when ON LAT

Role of microRNA

372 &373 = blok LATS & by following the HIPoo = to prevent

microRNAs and Signalling Gradients

• miRNAs can modify tissue responsiveness over time.
• Let-7 expression: increases during differentiation, resulting in progressive inhibition of RAS signalling.
• Signalling downstream of receptor tyrosine kinases (RTKs): involves two main branches, name the phosphoinositide 3-kinase (PI3K)–AKT and the RAF-mitogen-activated protein kinase (MAPK) cascades leading a aenereal up regulation Examples of the previous like, miR is is inhibiting mutlple nodes in multiple funtion & approach
• MicroRNA also can present with a pathway approach

miRNA and Networks

• In a cell, tf= inhibits/mirNA in turn inhibits the
• 1: A and is default where mir dominantes & f = 0 off of MIRNA (tf
• 2 in B extrinsic cue (signal with def) is repressed the MIRA that out comes

Example of MiR-200 Family

• 1 & 2 transcription the switch. Signal
• Transforming epithenlial is the to stabilizing expression

MicroRNA and Cancer Info

= over expross in lugn. & over in breast & linked to breast cancer & supresor in Breast but over in leukemia

How to Check Which Miro Is Importnain Cancer

• use cancer model:
• comparin miRNA experssion =profiling
• NGS = upregulating

MIR in Brana Cancel

• Genomic screens
• sequencing involved in medicate.

MicroRNAs as Tumour Suppressors

by blocking ocogenes & Mapping

• miRNA has the act cancer.
• 1 st was identified supresors delated in cronic lymphona. 1516 weredowun=
• Several suppressor w let-7 & 34 fams
• suppressors= by target

###MicroRNAs act Tumour suppressor

• target apoptosis.
• KRas-MIC the 7 of genes and by targating p3 cell.

###Miro as Onogenomes - Promote cancer genes

• act cancer gnes 3 years later or after tumor rep and 21. Reveal cartenogencis
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