Lecture #12 - Noncoding and Regulatory RNAs Flashcards
What can regulatory RNAs do
Overall – Regulatory RNAs can do eveyrthing that proteins can do
Primary function - Regulate gene expression
Ways Regulatory RNAs can functions
- 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 - Have RNA that binds to another RNA –> Changes the RNA structure
- RNA that can bind and can tether a protein and bring other functionality to a complex
Bacteria non-coding RNAs
Bacteria have variety of non-coding RNAs that play regulatory roles
Includes – tmRNA + ribozymes + RyhB
What are non-coding RNAs
Non-coding are RNAs are typically ‘genes’ expressed by the usual pathways in the cell (expressed using transcription and processing)
Eukytotic non-coding RNAs
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
tmRNA (overall)
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
Discovery of tmRNA
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
How does tmRNA fold
tmRNA folds to look like tRNA (has a CAA that can be charged with an alanine amino acid
How does tmRNA funtion
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
Typical action of a small RNA in bacteria
Overall - to regulate metabolic pathways
Example -RyhB
RyhB
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
Riboswitches
Bind to small molecules and regulate downstream events (cis regultory fcators)
Discovery of Riboswitches
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
B12 ribsoswitch
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
What are riboswitches involved in
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
RNA as an enzyme
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
When is RNA transesterification recation important
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
Eukryotic small RNAs (overall)
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
Where doe Eukryotc small RNAs derive from
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
Evolution of miRNA
miRNA = evolve more recently
miRNA is in more complex organism (complex organism have more miRNA and the miRNA plays more roles
Discovery of miRNA
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
Immunoblot of lin4 and lin14
Immunoblot shows that lin 14 protein in WT is found earlier in development and then absent in later development
Main Discovery with lin4 and lin14 to know they were connected
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
lin4 and lin14 relationship
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)
Sequences of lin14 and line 4
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
lin4 (overall)
Overall – Lin4 was the first discovered miRNA
Post discovery of lin4
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)
Let 7 sequences
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
Features of miRNA
- Endoed as a gene (use pol2)
- miRNA are dsicecrete genes
- Folded into hairpin strcture
3 . Well conserved
How many miRNA are there
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)
Discovery of many miRNA
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
How many genes can a miRNA regulate
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
Conservation of miRNA
Charts – shows parts of the miRNA that are conserved
Shows miRNA are generally conserved across species
- miRNA can have tissue specific expression
How are miRNAs produced (overall)
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
RNAse 3 enyzmes used for miRNA processing
- DROSHA (in the nucleus) –> DROSHA = cleaves the hairpins (cleaves near base of structure and makes a hairpin like structure)
- DICER in cytoplasm)
End of processing = yields 2-+ duplexes with 2 nucleotide overhands
How are miRNA made/Processed (overall)
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
RNAse 3 eznymes
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
Drosha
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
DICER
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
Helices portion of DIECR
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
miRNA processing steps
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
Guide strand vs. Passnager strand
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
2nd thing that DICER acts on
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
Fragemnts made by endogenous dsRNA vs exogenous dsRNA
Exogenous = makes fragments that are perfectlet complementary (complete complenets = fully bound
Endogenous = produces miRNA duplexes that have bumps and bulges (imperfectly complementary)
Discovery of RNAi + siRNA
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))
Sense vs. Antisense strand affect on transgene induced gene silencing
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)
What is the silencing trigger in transgene induced gene silencing
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
Recapitulation of RNAi in vitro
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)
What happened once made in vitro systems to show dsRNA affects gene expression
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
siRNA
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
How do the siRNA (miRNA?) function to brig about regulation of gene expression
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
shRNA vs. siRNA
SAME THING
shRNAs (put in as synthetic RNAs) –> enters the same complex that process the endogenous miRNA genes
Effector molecules in siRNA/miRNA processig (discovery)
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)
AGO proteins
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
X ray stricture of AGO architectire
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
How do dsRNA get sorted on AGO (miRNA)
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
How do dsRNA get sorted on AGO (siRNA)
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
AGO confirmation changes
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
miRNA and siRNA targets
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)
How does miRNA idetify targets
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)
Challenge in miRNA target identification
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
How do mamalian miRNAs affect gene expression
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
Is extnded pairing common/effective
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
How do siRNA silence gene expression
siRNA target enodnucleaic cleavage between nucleotides 10 and 11 of the siRNA then have decay –> THEN have standrad decay pathways
Specialization of small RNA pathways
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
Ciruclar RNAs
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)
Example of circular RNA
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
Processing of piRNA
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
What do piRNAs do?
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
What do we know anout hcRNA (heterochromatin RNAs)
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)
CRIPSR
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
Genome editting with CRISPR
