Lecture #12 - Noncoding and Regulatory RNAs

Question 1

What can regulatory RNAs do

Answer 1

Overall – Regulatory RNAs can do eveyrthing that proteins can do

Primary function - Regulate gene expression

Question 2

Ways Regulatory RNAs can functions

Answer 2

1. Have regulatory RNA that binds to target and blocks protein binding to the target mRNA
   - Ex. Changes the expresson of gene if binds to the mRNA of that gene
2. Have RNA that binds to another RNA –> Changes the RNA structure
3. RNA that can bind and can tether a protein and bring other functionality to a complex

Question 3

Bacteria non-coding RNAs

Answer 3

Bacteria have variety of non-coding RNAs that play regulatory roles

Includes – tmRNA + ribozymes + RyhB

Question 4

What are non-coding RNAs

Answer 4

Non-coding are RNAs are typically ‘genes’ expressed by the usual pathways in the cell (expressed using transcription and processing)

Question 5

Eukytotic non-coding RNAs

Answer 5

Eukaryotes have short and long ncRNAs

Eukaryotes have several classes of RNAs that are 20-30 nt in length (all play diverse roles in the cell
- Includes many classes of short ncRNAs –> miRNAs + siRNA s+ piRNAs + tsRNAs
- Includes long non-coding RNAs (many lnvRNAs made in transcription)
- Don’t know if all of the lncRNAs have function but we know some have function

Question 6

tmRNA (overall)

Answer 6

Overall - rescues ribosomes that are stalled on “non-stop” codons (need to get the ribosome off the mRNA)
- Function = ribosome recyling

tmRNA preforms the function of tRNA and mRNA

tmRNA = only in bacteria BUT eukaryotes also have pathways for recycling

Question 7

Discovery of tmRNA

Answer 7

Start - TmRNA was isolated for its weight in a screen and was catalogues as a gene

A separate group was trying to express mammalian genes in bacteria –> they kept getting small non-full length products –> they sequenced the truncated products –> found that at the end of all of the peptides was the same 11 amino acid sequence –> FOUND that the sequence of tmRNA corresponded to the sequences on the small peptides

After finding the 11 Amino acid sequence corssponded to tmRANA they use an RNA folding program on the tmRNA –> when they did this they found something in the tmRNA that looked like a tRNA

Thought maybe the tmRNA was playing a role of putting a tag on the end of the small protein products and could be fulfilling the role of recycling the ribosomes

Question 8

How does tmRNA fold

Answer 8

tmRNA folds to look like tRNA (has a CAA that can be charged with an alanine amino acid

Question 9

How does tmRNA funtion

Answer 9

tmRNA = rescues the ribsomes off the mRNA AND degrades the small protein that is made

Way tmRNA works – when have a staled ribosome on a nonstop codon the tRNA on the tmRNA can be inserted into the ribosome –> the peptide is transferred onto the Alanine that the tmRNA was bound to –> ribosome reads until the stop on the ORF on the tmRNA
- tmRNA does not have a start codon but it does have a stop codon = allows the ribsome to come off

tmRNA ALSO tags a degradation sequence at the end of the proteins = can degrade the small proteins made

Question 10

Typical action of a small RNA in bacteria

Answer 10

Overall - to regulate metabolic pathways

Example -RyhB

Question 11

RyhB

Answer 11

90 nt small RNA –> regulates a series of transcripts involved in Iron usage and metabolism

Functions:
1. Blocks ribosome binding (obstruction) and
2. Recuritmwnt function
BOTH can happens to the same RNA

Low Iron = RynB binds to an upstream area on the transcript –> causes an obstruction –> ribosome can’t bind –> have decreased ribsome binding

ALSO in low iron - RyhB can recruits RNAses that cleaves the transcripts = stop making the transcripts when don’t need

Question 12

Riboswitches

Answer 12

Bind to small molecules and regulate downstream events (cis regultory fcators)

Question 13

Discovery of Riboswitches

Answer 13

After PCR was made people were able to make RNA libraries –> People started doing experiments that showed that RNA was able to bind to things –> Found that that RNA could be enriched and that RNA could be bound to different things –> made people think that if RNA does this in vitro then maybe they are also doing this in a cell

TECHNICALLL 10 years before the discovery the RNA can bind to things – people found ribsowicthes but no one knew how it was working

Finally understood what a ribsoswicth was when they discovered the B12 riboswitch

Question 14

B12 ribsoswitch

Answer 14

B12 riboswitch = has a two portions of an upstream leader sequence (before the coding portion of gene) –> upstream leads sequence is folded when not bound (Green 1 and 2 portions bound in non-bound image) –> in this state the transcript can be tranlsated

The B12 riboswitch can ALSO form a aptamer domain when a metabolite binds to the riboswitch (Adpatmer domain binds to the metabolite) –> binding of the metabolite rearenges the structure of the upstream leader sequence –> Prevents the ribosome from binding –> prevents translation
- When have B12 = won’t get translation of genes that make B12

Question 15

What are riboswitches involved in

Answer 15

Riboswitches tend to be involoved in sensing of metabolite pwathways

To look for riboswitches –> People looked for instances where there is no known protein BUT know that there is metabolire regulting the productions of enzymes and the tranascirpts in its own pathways

Question 16

RNA as an enzyme

Answer 16

Overall – RNA can fucntiion as an enzyme

Before – people thought that only proteins function as enzymes BUT now we know that RNA can catalyze self cleavage
- RNA can have cis regulatory actions

RNA does a trans esterification reaction –> OH groups can carry out attack and cause cleavage of the RNA (endo-nucleaolystic self cleavage)

Example – Hammerhead ribozyme

Question 17

When is RNA transesterification recation important

Answer 17

Reaction is very important when have circular RNAs that need to be cleaved

Bacteria DNA is circular –> bacteria make a lot of circular transcripts –> these tyoes if ribsoxzymes (that catylyze self cleavge) are very useful in this context of bacteia making circular RNAs

Question 18

Eukryotic small RNAs (overall)

Answer 18

Eukryotic small ncRNAs includes miRNA + siRNA + piRNA
- miRNA –> regulates tranlsation + promotes RNA decay
- siRNA = cleaves traget RNA
piRNA = down regulates transcription + post-transcrtional gene silenceing

Question 19

Where doe Eukryotc small RNAs derive from

Answer 19

Overall - Come from genes

Genes makes the same miRNA every time vs. SiRNA is introduced as a synthetic tool and can get different RNAs at different times

Question 20

Evolution of miRNA

Answer 20

miRNA = evolve more recently

miRNA is in more complex organism (complex organism have more miRNA and the miRNA plays more roles

Question 21

Discovery of miRNA

Answer 21

miRNA and siRNA were co-discovere din the 90s but the people working on them didn’t realize that they were working on the same thing (didn’t realize until the biochemical mechanism was worked out)
- Early parts of the discovery were found using C.elegans (genetic based discoveries)

Discovery:
Ruvken and Ambros were looking at development in C.elgans –> working on the lin genes (looking at how mutation in genes affects development
- Gary Ruvken = was working on lin 14 – his lab fond that lin 14 was encoding a protein whose expression decreased during development
- At the same time – Victor ambros was working on lIn4

Found that lin4 phenocopied lin14 in many ways (lin14 gain of function and lin4 loss of function BOTH disrupt development by inhibiting terminal differentiation and causing recapitulation of earlier phenotypes –> Ruvkun and Ambros saw that they were seeing the same phenotype –> Gary ruvkin shared the seqence for lin14 with abros

Question 22

Immunoblot of lin4 and lin14

Answer 22

Immunoblot shows that lin 14 protein in WT is found earlier in development and then absent in later development

Question 23

Main Discovery with lin4 and lin14 to know they were connected

Answer 23

Found that a mutation in lin 4 could cause lin 14 protein to not be down regualted = produces the same effect as a gain of function in lin14 (loss of function in lin4 gives same phenotype as gain of function in lin14

Abros found that lin4 didn’ encode for any obvious ORF

Question 24

lin4 and lin14 relationship

Answer 24

Found that lin14 and lin4 lineage mutants phenocopies one another

ALSO FOUND Lin14 encoded a protein whose expression varied during development (high in L1 and low in L2/L3)

FOUND lin 4 had no obvious ORF but appeared to down regulate lin14 expression (negative regulator)

Question 25

Sequences of lin14 and line 4

Answer 25

Once compared the sequences of lin4 and lin14 Did northern blot --> found that lin4 RNA (doesn’t code for protein) was unregulated at the right time to be involved with the developmental transition After 3’ UTR sequences – Ruvkin found mutants (had a deletion that produced a mutant phenotype) ; Ambrose found that his ncRNA was paring with something in the 3’UTR in lin14 to impact protein expression Based on the sequences - looked at ways that lin4 and lin14 can bind - Don’t know which is actually hapepning but showed the idea that a small ncRNA might pair with an mRNA to influence mRNA translation

Question 26

lin4 (overall)

Answer 26

Overall – Lin4 was the first discovered miRNA

Question 27

Post discovery of lin4

Answer 27

AFTER discovery of lin4 in worms --> they found that small RNAs are conserved worm worms to humans - Ravkin found broader examples of small RNAs 2nd miRNA discovered was let7 - Found that in zebrafish let7 regulated developmental timing AFTER discovery of let7 the field exploded (people realized that these regulate gene expression)

Question 28

Let 7 sequences

Answer 28

Sequences (grey box shows the miRNA sequence) --> see that the sequence is identical across species (highly conserved) AND the binding site in the mRNA is ALSO very similar in different species (miRNA and the mRNA binding site are both similar in different species) Small ncrNA are in high abudnece as you go up the evolutionary tree

Question 29

Features of miRNA

Answer 29

1. Endoed as a gene (use pol2) - miRNA are dsicecrete genes 2. Folded into hairpin strcture 3 . Well conserved

Question 30

How many miRNA are there

Answer 30

Humans have >2500 ; dropshilla have 466 ; C. elegans have 434 - 271 organism have miRNAs There are MANY documents miRNA BUT we don’t really know the true amount - NOT all of the miRNA discovered are high confidence that they are actually a miRMA (might say they are an miRNA at first but then decide it is not)

Question 31

Discovery of many miRNA

Answer 31

Many miRNA were discovered by dave bartell lab - Batell lab = catalogued many miRNA --> came up with target scan that looks for bidning site for miRNA and sees how widespread they are Can look at which miRNA is actually functioning by looking at processing

Question 32

How many genes can a miRNA regulate

Answer 32

miRNAs are made from 1 gene BUT the miRNA can regulate many other genes - The binding site that miRNA bind to is small = many genes can be regulated by the same miRNA (because the gene sequence that the miRNA binds to is common) + 1 gene can be regulated by multiple miRNA - Each miRNA has many potential targets

Question 33

Conservation of miRNA

Answer 33

Charts – shows parts of the miRNA that are conserved Shows miRNA are generally conserved across species - miRNA can have tissue specific expression

Question 34

How are miRNAs produced (overall)

Answer 34

MiRNA are typically transcribed by RNA polymerase 2 = have a 5’ cap + polyA tail + can contain introns - miRNA can sometimes be found within introns (Called mitrons) - miRNA are often found in clusters miRNA processing – invloves two sequential RNAse 3 enzymes - End of processing = yeilds 2-+ duplexes with 2 nucleotide overhands

Question 35

RNAse 3 enyzmes used for miRNA processing

Answer 35

1. DROSHA (in the nucleus) --> DROSHA = cleaves the hairpins (cleaves near base of structure and makes a hairpin like structure) 2. DICER in cytoplasm) End of processing = yields 2-+ duplexes with 2 nucleotide overhands

Question 36

How are miRNA made/Processed (overall)

Answer 36

Pri-miRNA (Primary transcrtipt) is made in the nucleaus using RNA polymerase 2 (often occur in clusters that make a lot of pri-miRNA) --> After the cluster is made it is processed by DROSHA in the nucleus --> END with 20+ miRNA duplexes with 2 nucleotide 3’ overhands Clusters allow transcription to be co-regulated even though the downstream seps may be differentially regulated IMAGE - shows what the precursor look like

Question 37

RNAse 3 eznymes

Answer 37

RNAse 3 enzymes are ubiquitously involved in RNA processing (Called "molecular Rulers) Classes of RNAse 3 enzymes : 1. Class 1 = more simple than DROSHA or DICER 2. Class 2 = Drosha 3. Class 3 = Dicer Image - Right image = structure of DICER (has dsRNA in green) ; Middle = shows how the enzymes and RNA is positioned

Question 38

Drosha

Answer 38

Types of RNAse 3 proteins Drosha = has dsRNA binding domain + has 2 RNAse 3 domains Can only do 1 processing step of Pri-miRNA to forms the next precursor miRNA (forms a hairpin structure) that will be exported to the cytoplasm - DROSHA = forms a complex with cofactor DGCR8 (BOTH function in nucleus) Function - DROSHA + DGCR8 – pin the pri-miRNA (structure with many clusters) --> cleave the precursor pre-miRNA that will go to the cytoplasm --> in cytoplasm the precursor Pri - miRNA will be processed by DICER

Question 39

DICER

Answer 39

Has 2 RNAse 3 domains + dsRNA binding domain + helicase + PAZ domain DICER = functions in teh cytoplasm Uses the PAZ domain toanchor DICER to attach onto the 3’ overhand end of the pre-miRNA = positions it --> THEN DICER can move and cleave every 20 nt as it moves up the dsRNA

Question 40

Helices portion of DIECR

Answer 40

Helicase = important because DICER can process dsRNA in repetitive manner (DICER can cleave then unwind RNA and cleave again) RESULT – get production of processive fragments

Question 41

miRNA processing steps

Answer 41

NOTE - only shows 1 pri-miRNA but there could be many hairpins coming off Processing – Pri-miRNA is transcriibed using RNA polymerase 2 --> RNAse 3 DROSHA cleaves the pri-miRNA and makes a pre-miRNA --> pre-miRNA is exported by exprotin from the nucleus to the cytoplasm --> pre-miRNA is acted upon by DICER and its co-factor TRBP2 --> processing by DICER makes the miRNA duplex --> Duplexes will be loaded into appropriate Argoniate (AGO) to bring about gene regulation

Question 42

Guide strand vs. Passnager strand

Answer 42

Guide strand = strand that will be retained to function to repress the target (5p) Passenger strand = strand that will be released (3p) Which strand is the guide or the passenger can vary in different tissues or in a developmental dependent manner

Question 43

2nd thing that DICER acts on

Answer 43

DIECR = also acts on dsRNA that comes from exogenous sources - Example - DICER acts on dsrNA that is given to cells in lab OR acts on dsRNA injected by a virus

Question 44

Fragemnts made by endogenous dsRNA vs exogenous dsRNA

Answer 44

Exogenous = makes fragments that are perfectlet complementary (complete complenets = fully bound Endogenous = produces miRNA duplexes that have bumps and bulges (imperfectly complementary)

Question 45

Discovery of RNAi + siRNA

Answer 45

RNAi + siRNA = discvered in plants and neurospora How did they discover – reserachers were trying to produce petinia that would be more purple --> To make the petunias more pruple they took the gene they thought made plants purple and tried to over express the gene BUT they found that this actually decreased the purple color Overall – found that when have a transgene expressed there is a down regulation of endogenous RNA (Seen down regulation in gel (image))

Question 46

Transgene induced gene silencing

Answer 46

In plants = called co-supression In nuerospora = called quelling

Question 47

Mechanism for Transgene induced gene silencing

Answer 47

dDiscovered by Andy group by analyzing an already published paper --> When they read the paper they realized this this phenotypes required dsRNA

Question 48

Sense vs. Antisense strand affect on transgene induced gene silencing

Answer 48

People had thought that putting in RNA would act in antisense fashion (thought this would be why they got down regulation when added the transgene) BUT Ken found that he would get the same phenotypes whether he put in the antisense or the sense strand (confused scientosts) THEN Andy found that the biochemical method of preportion used for sense/antisense experiments meant there was ALWYAS a contaminating dsRNA --> thought it was the dsRNA that was needed (don't need antisense or sense alone)

Question 49

What is the silencing trigger in transgene induced gene silencing

Answer 49

found that the dsRNA was the silencing trigger HOW - Andy found a way to put the sense and antisense together (dsRNA) OR just the sense OR just the antisense - Readout = visable phenotypes (twicthing + hermaphridites) When add antisense or sense analong ONLY = get WT BUT when add both teh antisen and the sense (dsRNA) get the mutant phenotype - Realized that if they target the intron with sense and antisense together (dsRNA) = get WT --> prelude to the fact that this process occurs in the cytoplasm = doesn’t affect intron

Question 50

Recapitulation of RNAi in vitro

Answer 50

Key breakthrough of RNAi in vitro using drosphila empbyroes or Drophila S2 cells or HeLA cells - Drosophilla embryo lyaste + dsRNA = specifc degredation of traget KNEW - dsRNA affacects gene expression from protein coding RNAs --> To understand the mechanism they recapitulated findings in an in virto system --> reconstitute the system --> Showed that when put in dsRNA in vitro --> have down regulation of target RNA - Control RNA is unchanged in gel = shows this has specificity - Can look at this over the course of time (seen in gel)

Question 51

What happened once made in vitro systems to show dsRNA affects gene expression

Answer 51

Once made in vitro system they were able to isolate what kinds of complexes are doing this - Could see that small RNA that are made (even if they put in longer transcripts those transcripts still get made to smaller pieces) - Found that a longer RNA that people put in t0 do antisense strategies are being processed to smaller dsRNA (small dsRNA is needed for this effect) - Saw phenomon in co-supression in plants + in in vitro extract NOW know - DIECR = make the small dsRNA in the cytoplasm

Question 52

DICER

Answer 52

Function – processes the long dsRNA - Can put in a long dsRNA and get smaller dsRNA (Can put in long dsRNA or a short dsRNA and get the same outcome) - People can also put in siRNA based hairpin --> put in dsRNA in way that form that looks like the miRNA precursor and still get the same sort dsRNA that functions

Question 53

dsRNA that are put in my people (exogenous dsRNA)

Answer 53

ALL lead to siRNA (not genes – siRNA is put in synthetically) dsRNA that people put in are perfectly complentray to each other and are then cleave to siRNA by DICER TRBP - siRNA doesn’t have the bulges that miRNA has - Because not made from a gene and processile processed by DICER = can get diferent siRNA

Question 54

siRNA

Answer 54

Most siRNAs refers to exogenously introduced RNA that feeds into the endogenous RNAi system…but dsRNA can also be generated in the cell (endogenously) siRNA = not genes (siRNA is put in synthetically) Can be transient or stably expressed DICER will prccess the duplex in the cytoplasm ALL versions lead to generation of siRNAs

Question 55

How do the siRNA (miRNA?) function to brig about regulation of gene expression

Answer 55

Mammals – pri-miRNA is made (Capped with the PolyA tail) --> DROSHA cleaves pri-miRNA to make the pre-miRNA --> most of the time of the pre-miRNA is processed by DICER to miRNA duplex --> one of the strands from the dsRNA duplex will be incorportated onto a RISC complex by binding to AGO (loaded to AGO) --> leads to translational repression and mRNA cleavage - Most cases – translational repression occurs first then have mRNA cleavage BUT can be reverse

Question 56

How does siRNA (miRNA) leads to decrease expression

Answer 56

Most cases – translational repression occurs first then have mRNA cleavage BUT can be reverse BUT Can have the primary effect that impacts gene expression be the mRNA cleavge - Ex. In non-primary cells such as cancer cells) BUT that is not always the likes (not the case in primary cells like neurons ; can get translational repression in absensce of mRNA cleavage) In some instances have non-conical function – cleavage activity of AGO itself (NOT RISC) can cleave out the strand to be loaded to the to the RNA induced silencing complex (rare)

Question 57

DROSHA in siRNA processig

Answer 57

DROSHA = recognizes the structure of the bumps and bluges on miRNA = siRNA won't be processed by DROSHA

Question 58

shRNA vs. siRNA

Answer 58

SAME THING shRNAs (put in as synthetic RNAs) --> enters the same complex that process the endogenous miRNA genes

Question 59

Effector molecules in siRNA/miRNA processig (discovery)

Answer 59

To discover what the effector molecules are – need to purify and reconstitute the system in vitro (Used to understand how miRNA regulates gene expression) - Can use in vitro as a readout to isolate the complexes Experiment - Showed down regulation of targeted transcript without impacting control transcription - Found In Immunoblot = have AGO2 protein that is co-migrating with suppressive activity (shows AGO might be involved) - Saw AGO is necessary and that they are sufficient to generate substrate cleave in siRNA situation END - Can recapulate in vitro = get cleavge event (seen in gel in image)

Question 60

AGO proteins

Answer 60

AGO = found in all organisms - Very diverse family of proteins used in RNAi (found in plants + neurospora + C.elegans) ALL AGO proteins share a PIWI domain and a PAZ domain - PIWI domain looks like RNAse H domain structurally

Question 61

What do PAZ and PIWI domains do

Answer 61

PAZ domain = binds to the 3’ end of RNAse AGO PIWI domain = looks like RNAse H = PIWI domain in AGO functions like RNAse H - Function of PIWI = cleave where the miRNA or siRNA is bound to the target

Question 62

X ray stricture of AGO architectire

Answer 62

Structures show the PIWI domain + PAZ domain + Mid domain - Mid domain = important for binding the 5’ end of the miRNA - PIWI domain = involved in cleavage

Question 63

How do dsRNA get sorted on AGO

Answer 63

Question - How do you get 1 strand that is doing the function and the other strand ejected from AGO? ideas for how this can happen (BOTH use asymetric loading): 1. miRNA uses the bulges 2. siRNA uses PIWI domain

Question 64

How do dsRNA get sorted on AGO (miRNA)

Answer 64

Overall - Asymmetric loading because of the bumps and bulges on the miRNA --> Budlges may lead to a preferance for which strand can bind to PAZ and the MID pocket miRNA has more of a have distiction of which strand can be loaded than siRNA - often find that in a given tissue or developmental time that the primary miRNA functioning in this manner will only be 1 strand and the other strand is in lower abundence

Question 65

How do dsRNA get sorted on AGO (siRNA)

Answer 65

siRNA strands don’t have bumps = can’t lead to preferential retention of 1 strand BUT because they are identical they can trigger PIWI domain (PIWI = RNAse activity of AGO) to cleave them and release a passanger strand = only left with guide strand

Question 66

How does RISC assemble

Answer 66

Overall - RISC assembly from duplex RNAs When AGO is unloaded it is a closed confirmation --> THEN have chaparones (hsp70 and hsp90) that open AGO and allow the duplex RNA to enter into AGO --> one strand of the duplex THEN engages in binding to the PAZ and MID domains --> unwinding when the strand binds to PAZ and MID leads to the ejection of the other strand - Free small RNA strands are degraded = the second strand that is released will be degraded (unless participates in separate AGO) --> second strand will be present at lower levels in cell or tissue

Question 67

AGO confirmation changes

Answer 67

Right image – shows how AGO confirmation changes in response to binding events Confimation chnages = PAZ domain binds to the 3’ end of miRNA = causes a confirmation change that results in the ejection of the strand that didn’t bind

Question 68

Why does 1 strand binds preferentially over the other

Answer 68

There has been an effort to understand why 1 strand binds preferentially over the other Answer - Some people say that a weaker 5’ pairing dictates which strand will be involved (weaker 5’ pairing might be retained in AGO) but that doesn’t always seem to be true - Can change in different tissues/different stages of development

Question 69

miRNA and siRNA targets

Answer 69

miRNA - Binding of miRNA to imperfectly complementary targets leads to cleavage-independent silencing (get translation repression + sometimes decay) - no confirmation chnages to RNA = no cleavage siRNA - Binding of siRNAs to perfectly complementary targets leads to cleavage at positions 10-11 using PIWI domain - Have conformational change to RNA = results in cleavage using PIWI domains Different confirmations of RNAs on AGO protein in two situations could explain the different biochemical outcomes - Once have 1 strand loaded onto AGO --> AGO will pull in other proteins complex to form RNA induced splicing complex (RISC complex)

Question 70

miRNA vs. siRNA mRNA degredations

Answer 70

miRNA - IF mRNA is degraded it is degraded through decay pathways NOT nuclease pathways siRNA - mRNA is degraded by nuclease pathways

Question 71

How does miRNA idetify targets

Answer 71

Overall - miRNA use seed pairing to identify targets (seed then bulge then 3' pairing - Seed = nucleotides 2-8 on the 5’ end of the miRNA (red nucletodes in image) - 3’ binding occurs to different degrees - miRNA will scan the UTR of the mRNA and look for the seed side Image – shows lin4 and lin 14 have perfectly complementary seed binding (bind then have a bulge then have imperfcet complementary binding at the 3’ end)

Question 72

How prediction programs look for the target sequence (Ex. How does target scan work)

Answer 72

Take the seed sequence and scan the 3’ UTR of mRNA and look for seed site and then spit out potential targets that match to the miRNA that you are looking at - Prediction programs use complentarity + stability + conservation + accessibility of a site to determine strong candidates Issue - In C.elagans + mammals + drosophilla most miRNAs are imperfectly complementary to targets = makes it challenging to predict the target of miRNA

Question 73

How does miRNA regulate target

Answer 73

miRNA can regulate target transcripts by cleavage or translational repression

Question 74

Impact of miRNA

Answer 74

Impact of miRNA can be affected by availability of target or the abundance of miRNA If the miRNA has a good seed match for the targets BUT if the miRNA is in low abundance then the miRNA won’t have a huge impact on the translation of the mRNA target

Question 75

Ways decreased miRNA function can be compensated for

Answer 75

Ways decreased miRNA function can be compensated for: 1. Many 3’ UTR of the mRNA ALSO have a binding site for multiple miRNA = multiple miRNA can bind together to influence translation of the transcript - ALL miRNA will work together (Often have a small amount of regulation by each miRNA) - ALL miRNA regulate many transcripts = many human transcripts are regulated by miRNA at some point 2. Have some miRNA that are abundant = can carry out regulation on its own

Question 76

Challenge in miRNA target identification

Answer 76

Imperfect base pairing allows miRNAs to potentially interact with many targets THIS complicates human prediction of the mRNA that the miRNA targets ALSO seed region is small = means that when you run through a prediction program you get many targets Solution = attaching the ncRNA (miRNA) to the target RNA --> then use sequence to make libraries --> sequence interactions (tie miRNA and mRNA together and sequence) - Can be done in mamalian setting + in a cell type specific way

Question 77

Results of miRNA target identification

Answer 77

Experiments show that prediction of perfect seed match is NOT always the case --> INSTEAD can have a site where you don’t have a good seed match BUT miRNA can still bind to mRNA - Bad seed match can be compensated IF there is better matching in the 3’ of the miRNA Method ALSO shows that you can have interactions of miRNA with regions of the mRNA that is outside of the 3’ UTR

Question 78

3' UTR interactions of miRNAs

Answer 78

3’ UTR interactions is most standard miRNA interaction because those interactions would be more stale as the ribosome move son the transcript the miNA would be less likely to be kicked off the transcript and have more time to dwell there and recruit the other protein components that will affect the degradation or stability of the transcript

Question 79

How do mamalian miRNAs affect gene expression

Answer 79

miRNA = reduces gene expression using mRNA decayed Overall - RISC complex can inlude porteins that can decap or deadnylate --> ultimtley degrades the transcripts AGO recruits GW182 (GW is in RISC complxex) --> AGO binds to GW182 proteins --> GW proteins interact with the NOT complex --> NOT1 interacts with Ddx6 - NOT complex = invloved in deandylation) - Ddx6 = DEAD box helicase - GW182 = acts as a scafold to recuit RNA binding porteins and regulatory proteins that silences the complexes --> impacts tranlsation elongation or intaition

Question 80

How do plant miRNAs affect gene expression

Answer 80

Plant miRNA often have perfect complementary miRNA/mRNA binding (use a siRNA like mechanism = cleaving through PIWI domain)

Question 81

What drives the turnover of miRNA

Answer 81

miRNA stability varies between specific miRNA in the same cell and in different cellular contexts Turnover occurs by: 1. Tudor-SN (nucleuase) can cleave and downregulate certain miRNAs 2. Target directed miRNA degredation (TDMD)

Question 82

Target directed miRNA degradation (TDMD)

Answer 82

TDMD = mechansim for regulating miRNA abudnence Overall - Target causes destruction of the miRNA instead of the miRNA destorying the target - Uses miRNA +trigger RNA + E3 liages + proteomsome Extended miRNA binding sites that trigger TDMD have a seed match and a small bulge and extensive 3’ biding site (extended pairing = 6 nucleotides in the miRNA 3' end, lacking central pairing (nucleotides 9–11) - Extended miRNA binding site = found on snRNA and lncRNA and in the 3’ UTR where miRNA typically binds

Question 83

Options for what can happen when miRNA binds to target RNA

Answer 83

1. 99% of miRNA/target RNA interactions have seed pairing and inhibition of translation and dacaping and depolyA and decay 2. Rarely have full pairing and target RNA cleavage 3. Rarley can have extended pairing that can lead to degradation of the miRMA

Question 84

Is extnded pairing common/effective

Answer 84

Extending pairing is NOT a common interaction BUT it is effective meams for controling the level of miRNA because the trigger can be recycled Binidng recuites Zswim 8 (E3 ligase) --> leads to ubiquiniation and degredation of AGO --> miRNA is then unprotected and will be degrade by normal mechanism --> Trigger RNA can then go back and do the same extensive pairing again - Recruit Zswim 8 because have chnage in confrimation fo AGO when have extsnitive 5’ and 3’ complementary binding with a small non-binding regions in the middle

Question 85

How do siRNA silence gene expression

Answer 85

siRNA target enodnucleaic cleavage between nucleotides 10 and 11 of the siRNA then have decay --> THEN have standrad decay pathways

Question 86

Specialization of small RNA pathways

Answer 86

Mamals have specializaton of the small RNA BUT in drosophila the different AGO proteins preform different things In drosophilla - AGO 2 = does RNAi BUT AGO 1 = does miRNA mediate gene repression in mammales - AGO 1-4 all function in the same miRNA pathway - AGO 2 in vitro can slicing activity BUT ALL AGO can function in he miRNA pathway - When knowdown 1 AGO then a different AGo will just be upregulated and will complensate for function

Question 87

Ciruclar RNAs

Answer 87

Circular RNAs = made from back splicing of transcripts - aka Competing endogenous RNA Function: 1. Act as sponge for miRNA by having multiple miRNA biding sites + act as sponge for RNA binding proteins - impact gene expression of the target RNA that the miRNA or the RNA binding proteins bind to 2. Have sites that interacts with miRNA in target directed miRNA decay pathway (circular might engage in TDMD)

Question 88

Example of circular RNA

Answer 88

Example circular RNA = sir 7 Function - Sir7 binds to mir7 --> prevents mir7 from repressing its target - allows the target of mir7 to under go translation

Question 89

tRNA-derived small RNAs

Answer 89

tRNA-derived small RNAs = source of ncRNA that comes from fragments like tRNA or rRNA Includes - tRNA-dervived fragemnts (tRFs) and tRNA halves (tiRNAs) + cleaving rRNA and to get ncRNA fragemnts - tRFS = can bind to RNAs or bind to RNA binding proteins Because the abudnce of rRNA and tRNA is so high even if some fragments have a biological significance then it will likely have functional effect

Question 90

Abundence of small ncRNA types

Answer 90

miRNA = most abundent tRFS = second in abudnce

Question 91

tRFs

Answer 91

14-32 base single-stranded RNA derived from mature or precursor tRNAs Distinct from the stress-induced tRNA fragments created by cleavage in the anti-codon loop Function - functions range from RNA silencing to priming a retroviral reverse transcriptase

Question 92

Gurdians of the genome

Answer 92

piRNA

Question 93

piRNA

Answer 93

Longer than siRNA or miRNAs (up to 33nt) Location - in nuclues (Mostly found in gonadal cells) - Important in gonads in keeping regions of the genome silent Can be transcribed from repetitive sequences in the genome (retrotransposons, DNA transposons, microsatellites) Function - downreguates transcription or transcripsts from repetitive genomic regions (Ex. feed back to interfere with gene expression from the loci piRNA is made from) - Functions with PIWI - Look and function like heterochromatic RNAs (in yeast)

Question 94

RNAs down-regulate transcription from diverse repetitive regions

Answer 94

hcRNAs (heterochromatic) from S. pombe piRNAs (Piwi-interacting) from mammals

Question 95

What do we know about piRNAs

Answer 95

Derived as long single stranded transcripts from genetic loci (genetic loci = piRNA clusters) - Found in transposon-rich areas of the genome piRNA have Dicer-independent processing piRNA is loaded directly onto Argonaute protein (PIWI) as single stranded species

Question 96

Processing of piRNA

Answer 96

piRNA = Produced from repetitive regions in the genome Undergo ‘ping pong processing’ --> have primary cleavage events that allows ncRNA to be loaded to PIWI protein --> once loaded the piRNA can target the primary transcripts to make more piRNA - Ping pong model = repetitive model to keep making more piRNA - Leads to generation of a lot of piRNA that can feedback and can silence transcripts that come from repetitive regions of DNA

Question 97

What do piRNAs do?

Answer 97

PiRNA = Important in germline and during early development; silence expression from repetitive regions that could be problematic at this sensitive stage - Silences epxression from regions that you need to keep silent to maintain pluripotencey or to maintain differentiation or maintain identity of early cells Function: 1. Like a canonical siRNA that cleaves nascent transcripts (cleaves in trans) 2. Through epigenetic changes in the DNA and chromatin structure

Question 98

What do we know anout hcRNA (heterochromatin RNAs)

Answer 98

hcRNAs = Help with integrity of the centromere - Function - silences expression and maintain chromatin in certain regions of the genome Bidirectional transcription yields duplex processed by Dicer endonuclease (SpDcr1) and loaded into an Argonaute (SpAgo1) RITS complex (containing SpAgo1, a chromodomain protein Chp1, and a GW-repeat protein TAS3) then targets nascent transcripts which recruits RdRP which synthesizes more transcripts RITS complex also triggers patterns of histone H3 lysine 9 methylation which broadly silences these regions (forming heterochromatin)

Question 99

Evolutionary origin of these pathways

Answer 99

Complex RNAi = last common ancestor --> required AGO/PIWI + DICER + RNA depent polymerase Likley was an ancetsral viral defense response (likey againt viruses and other genomic arasits like ranpsons)

Question 100

dsRNA in mammals

Answer 100

Mammals = don’t use long dsRNA ssrNA is not used in mammales like it is in other organisms – instead we have complex immune systems SO in mamales are infected with dsRNA it will mount an interferon response so we don’t get selective silence that you would in other cell types instead use immune system to fight off virus

Question 101

CRIPSR

Answer 101

CRISPR is a defense system in bacteria against bacterial phage (example on ncRNA function) Inlcudes 3 phases: 1. Insertion of sequence into genome 2. Transcription and processing of CRISPR locus 3. Targeting of foreign DNA Three major subtypes: 1. Cascade (type I) - structures 2. Cas9 (type II) – engineering

Question 102

Genome editting with CRISPR

Answer 102

Question 103

Why is CRISPR better than RNAi

Answer 103

1. Engineer of lock NOT just knockdown 2. More effcicnet than HR 3. Works broadly with few components

Question 104

Long non-coding RNAs

Answer 104

LncRNAs = look like POl2 transcripts (spliced + have 5’ CAP + polA tail) BUT have limited protein coding potential MOST lncRNAs have uncharactreized function but a few have regulatory functon of protein coding gene Best charcterized = Xist Emerging example of lncRNA = participates in TDMD that regulate the level of miRNA - Bind to miRNA and destroying the miRNA through TDMD

Question 105

Xist

Answer 105

Xist = 17kb (not very conserved sequence) Function - mechanism of gene dosage compensation Xist = expressed from inactive X chromsome --> coats the X chromosome and recuolts the plycome (PRC2) and silences the X chrosme through histone trimethylation