L35 MiRNAs Flashcards
miRNAs
- non-coding RNAs
- 20-22 nt form there is a 60nt hairpinlike precursor
- they bind to partially complementary sites
- repress translation and /or mRNA stability
- a single mRNA can target more than one mRNA
- a single mRNA can be targeted by MORE than one mRNA
lin-4/lin-14
- discovered in C-elegans
- this gene (although short and doesnt code protein), produces RNA products: lin-4L and lin4-S (for short and long)
- forms a hair-pin like structure
- the short one can base pair with lin-14
- the classical encoding gene
- makes sense becuase lin-14 is important for promoting stem cell proliferation while lin-4 is important for differentaition
- suggest lin-4 is a regressor for lin-14
- first generation of micro-RNAs that was discovered
(2000) let-7
- similar to lin-4
- difference is that let-7 is across multiple species
- forms a hairpin like structure
- let -7 is also evolutionary conserved: they found across many species
- let scientist to realize that this is present in many organisms
- also found let-7 can target more than one mRNA AND a single mRNA can be controlled by both let-7 and lin-4
The Canonical Pathway (biogenesisis of miRNA)
- begins in nucleus where genes are transcribed; typically quite long (this is called the primary microRNA transcript)
- once primed, first processing occurs within the NUCLEUS and involves excision of the step-loop element from the longer primed miRNA by a microprocessor (Drosha)
- stem-loop structure released
- loop exported to CYTOPLASM by the EXPORTIN-5/RAN-GTP
- second processing reaction is catalyzed by DICER which forms another protein complex
comes from riboneuclese - (chopping and dicing) occurs
- loading the miRNA into miRISC particularly forming contacts with argonaute is the last step
the sequence of which microRNA is transcribed from in the Nucleus
primary microRNA transcript (pri-mRNAs)
How are primary microRNA transcript (pri-mRNAs) produced?
- RNA polymerase II (Pol-II) (but sometimes can be by Pol-III)
- contain tails
- pri-miRNAs also have a tail
- longer sequence have a stem loop like structure that is destined to become the miRNA
- the rest of the structure is a waste (but sometimes they can encode many so its more useful)
- b contain intron and exons sometimes (here its shown by b (shows non-encoding and encoding areas depending on if they are introns or exons)
- can also be encoding of pri-MRNA in addition to pre-RNA shown in C - yellow means that those parts can encode protein
Pol-2
- RNA poylmerase 2
- encodes the pri-miRNAs
- but can often be impacted by tissue-specific transiption factors
(blue pigment shows miRNA expression)
first processing of miRNA occurs in the ______ and involves ___ by an enzymatic microprocessor complex called ____
Nucleus; excision of the stem loop of the miRNA; Drosha
exports the miRNA from nucleus to cytoplasm
exportin-5/RanGTP
second processing reaction is catalyzed by _____
Dicer
‘cropping’ and ‘dicing’
DROSHA is a nuclear RNAseIII-type endoribonuclease that crops the stem loop pre=miRNA element 9b) from pri-mRNA 9(a)
DICER is a cytoplasmic RNAseIII-type endoribonuclease that cuts off (“dices”)
the stem-loop from pre-miRNA to form a short RNA duplex (c) with 3’-protruding ends
the miRNA duplex
- only one strand is destined to become something (the ‘guide’ strand)
- there is also a passenger strand that is degraded normally
loading the miRNA into ________ particularly forming contacts with ________is the last step before it does its job
miRISC; argonaute
miRNAs as a part of RISC can now go and bind to the target mRNA or basepairings and control RNA stability and translation stability
- majority are negative though so they inhibit production of protein
dependent factor of miRNA activity
what argonaute subunit is present in the RISC (there are 4)
- similar to each other
- Ago2= physical clevage when perfectly base paired
Ago2 + Perfect Base pairing =
mRNA clevage and rapid decay
Any Ago (1-4) or mismatched base pairing
- most common to happen
-outcome: inhibition of translation - mRNA destabilization through deadenylation
Recruitment of ____ from miRNA that ______
CCR4-NOT (a deadenylatioin ezyme); shortens the 3 prime tail which immediately make the RNA more vulnerable and destabilized and it may also reduce translation efficiency
P-body
compartment in cytoplasma (not bound by membrane) that contains a number of RNAse degredagtion enzymes
- so its recuritment from miRNA is another mechanism that = RNA destabilization
The 2 mechanisms that miRNA use to destabilize RNA
- CCR4-NOT (to cut the 3 prime tail)
- P-body (cytoplasm comparment with degredation enzymes)
Stem cell maintence in mammals (normal bio role of miRNAs)
- help stem cells maintain proliferation status
- in diseases important (if this regulation is disrupted)
- miR-371 and 302 in humans
- have targets of cell cycle inhibitor targets of CDK inhibitor p21 and Lats
- these proteins put breaks on cell proliferation and miRNAs expressed in proliferating cells target these cell cycle repressors
- pluripotent stem cells self renew and diff. into diff types of cells so imp.
- but DONT ALWAYS promote, quite often can PROMOTE DIFFERENTIATION????
Neuron development in nematodes (normal bio role of miRNAs)
- Left and Right chemosensory neurons (ASEL or ASER) sense different chemicals
- to develop left need die1 and right need cog-1
- so to develop left CANT develop the other
- can only have either or, never both
- this is controlled by miRNAs- becuase die-1 targets lsy-6 producing the pri-miRNA for it
- in turn lsy-6 represses cog-1
- this means the die-1 can be expressed even more (look at chart)
- conversely, cog-1 miR-273 pir-miRNA is produced, etc the same loop but reversed
Deregulation of specific miRNAs (microRNA pathway in Cancer)
- similar to protein coding genes, miRNA can be oncogeneic or tumour supressive
- oncomeres
- oncogenic miRNAs: downregulate expression of tumor supressive coding genes and vise-verse
(stopped at 44:12)
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miR-17/19
- example of oncogenic miRNAs
- B-cell Lymphomas
- miR-19 is a regressor of a tumor supressor called PTEN so it is in turn is a repressor of pro-survivial kinase or PKB,
- consequence of that is that is htat the PKB is important for cancer to maintain the cell viability and if you downregulate it, it will lead to apoptosis
- so if you allow a lot of APT to be expressed (b/c now PTEN is inhibited my the microRNA); the cells survive
- so ability to resist apoptosis occurs- this is how miRNA enforce this
What allows for increased miRNA equally apoptosis resistance in Cancer?
this cluster (mi-17/mi-19) is upregulated by C-MYC (a transcription factor that allows the miRNA to gain its function) either by allow more miRNA copies themselves or by increasing the levels of c-MYC
testicular miRNAs _______ are _______ and = tumorigensesis because_________
miR=371/372/373; overexpressed; they inhibit a tumor supressor called LATS2 that works by blocking the activity of proliferative pathway (e.g. 1 that converges on CDK)
- this means that cells proliferate extensively
let-7 in cancer (as a tumor supressent)
- inhibits RAS and HMGA2 (which are oncogenic)
- when let-7 is downreg, those proteins are at higher concentration == more proliferation and cancer
miR-15/16
- tumor suppressor
- cluster that mutation or downregulated means that increases proliferation (bc increases cyclin-D and Wnt-3 signal molecule (which both promote cell proliferation) and therefore tumor progression
miRNA in Diagnostics
- in blue miRNA cluser, the BLUE is more which means that you can help see where cancer is originating easier
miRNA expression is often generaly _____ in tumors
repressed; as you can see by the more blue area in this diagram
- this means that they are liekly beneficial in protecting against tumors
3dicer with miRNA and cancer realtion
- low dicer is bad news for cancer progression
- it would == less miRNA because dicer is needed to make them
- this could be a potential treatment target
