Regulation by RNAs
How does the CRISPR-Cas System work?
-Phage is inserted into DNA -CRISPR Cas chops the DNA -Spacer is derived from the phage and is incorporated at the beginning of the CRISPR array -The CRISPR locus is transcribed and processed into short crRNAs with each crRNA associating with proteins to form a crRNP complex -The crRNP complex surveils DNAs in the cell, and cleaves any DNAs that are complemnetary to the spacer sequence in the crRNA
How many subtypes degrade RNA sequences, how many degrade DNA sequences?
1 Subtype (Type 3B) degrades RNA rather than DNA and 4 subtypes degrades DNA
What is the pathway for processing and DNA cleavage?
1) A precursor transcript is generated of the CRISPR array 2) Repeat sequences form hairpin structures 3) Transcript is cleaved into single units of one repeat and a spacer 4) The cascade complex forms on the crRNA with the Cas3 providing a DNAse activity (for the subtype shown) 5) DNAs in the cells that are complementary to the spacer RNA sequence are cleaved by Cas3
What are the 2 general modes of action via srnas?
1) Activiation/repression of translation 2) mRNA degradation
What are the 2 parts of the riboswitch?
1) Aptamer - RNA that binds the metabolite that has a secondary RNA structure at the 5' end - an RNA "anti-body_ 2) Expression platform - contains regulator sequence for transcription (i.e. RBS or Terminator) downsteam of the aptamer region
What are the 4 consequences of activated RISC?
1) Degradation of complimentary RNAs: argonaute (slicer) cleaves RNA complementary to the guide RNA and ususally happens when the match is perfect 2) Translational inhibition of complementary RNAs that ususally happens when the match is imperfect 3) In the nucleus, complementary genomic sequences are inactivated by chormatin remodelling enzymes and prevents transcription 4) Amplificiation (only in some organisms) an RNA-dependent RNA polymerase (RdRP) copies mRNAs into dsRNA
What are the 3 domains of a dicer and its function?
1) PAZ domain that ancours the 3' end of the dsRNA 2) Handle domain - spacer region that controls the length of the RNA 3) Blade domain- contains the active site (2 metal ions) that can each cleave 1 end of the RNA and produce a 3' end that can fit into the Paz Domain of another dicer
How domains does argonaute protein have?
1) Paz Domain that recognizes the silencing RNA 2) RNase domain cleaves target RNAs that are complementary (cleavage of sRNA) opposite of the guide RNA
What are the 2 mechanisms for regulation of RNA in eukaryotes?
1) RNAi 2) Long non-coding RNAs (Inc RNAs; lincRNAs)
What are 3 ways in which regulation by RNAs occur in bacteria?
1) Small RNAs (sRNAs) 2) Riboswitches 3) CRISPR-Cas System (not really regulation but has similarities to regulatory RNA)
Describe the process of RITS?
1) Transcrips of repeats hybridize to each other and are processed by Dicer into RITS complexes 2) The RNAs in RITS pairs with complementary RNAs in the cell, and recruit chromatin-remodelling complex to methylate and silence the repeat DNAs 3) These siRNAs are known as piRNAs or PIWI-interacting RNAs
What occurs with RNAi regulation?
1) dsRNA is introduced into a eukaryotic cell or is produced in the nucleus via miRNA 2) Dicer cuts the dsRNA into pieces of 21-23 bp which are now called siRNAs or small interfering RNAs 3) The dsRNA is incorporated into RISC (RNA-induced silencing complex) with a passenger RNA and a guide RNA 4) The passenger strand is cleaved and leaves the guide RNA -> activating the RISC complex 5) Can act in 4 different pathways
What is the mechanism for siRNA amplification?
1) siRNA-RISC complex forms and dicer cleaves 2) the complex binds to targeted mRNA 3) Polymerase reaction that occurs when RdRP elongates the siRNA in a 5' to 3' direction 4) Dicer cleaves the dsRNA that is polymerized to produce multiple siRNAs via the cleavage of a dicer 5) Process can be repeated in amplificaiton
What does the spacer region correspond to?
30 bp sequences derived from phages and plasmids
What bp length do miRNAs target?
8-10 bps
What is the main mechanism sRNAs use for regulation?
Activiation/repression of translation
How is CRISPR-Cas used to create a site specific mutation?
Along with the crRNP, a dsDNA with the desired sequence mutation is introduced into the cell. Process initiated by a DSB in viva. Homology-directed repair introduces the sequence of the repair template into the genomic sequence
What does dicer and drosha cleave?
Dicer cleaves one side of the transcript first and Drosha cleaves the other strand for a DSB that is asymmetric
Are miRNAs difficult to identify and why?
Difficult to identify and evaluate all miRNAs because their sequences are short and many are "hiddne" within gene sequences
What two enzymes process miRNA?
Drosha and Dicer
What is the PAM also recognized in?
During subsequent surveillance and cleavage reaction, the PAM sequence is also recognized in the target DNA sequence by the nuclease
What genes are variable across subtypes?
Genes that are involved in crRNA processing, crRNP formation and target degradation are variable (and especially proteins in the crRNP complex)
What is the regulatory RNA in eukaryotes?
IncRNAs
How does ligand binding regulate translation?
Ligand binding can regulate translation by hiding or exposing the shine-dalgarno (RBS) sequence
Does siRNA amplification occur in humans?
No
What is the PAM sequence?
Prospacer adjacent motif that is a DNA sequence of approx 3 bp that is recognized by protein during the acquisition step
RITS?
RNA-induced transcriptional silencing
What are the regulatory RNAs in eukaryotes?
RNAi
Function of miRNA in biotechnology?
RNAi makes it easy to experimentally disrupt gene expression in vivo. A short hairpin RNA (shRNA) is expressed in vivo corresponding to the sequence of a desired gene. The effect is a "knock-down" rather than a "knock out" so it is amenable for essential genes (i.e. cells have the gene but the amount of expression is greatly decreased and done with essential genes)
what family is a dicer part of?
RNase 3 family enzyme (specific to dsRNA)
What are the components of CRISPR-Cas?
Repeat, spacer and repeat
What organisms are riboswitches found in?
Riboswitches are found mainly in bacteria but there are some examples in eukaryotes
What is sam?
S-adenosyl methionine, a common ligand for riboswitches, with the riboswitches having 2 different expression platforms
In what regions does silencing occur for the RNAi pathway?
Silencing of repeat regions occurs through the RNAi pathway
How is CRISPR-Cas used for genome engineering?
Since Cas9 is the only protein in the crRNP it makes it easy to engineer so that there is 1 RNA transcript instead of 2- making it simple. During surveillance, a sequence of approx 30 bp is recognized for cleavage, including the 3bp protein recognition(PAM) and 30 bp RNA recognition (spacer RNA-DNA pairing) This causes the activation of nuclease domains and targeted degradation by non-cas DNases
How does activiation using sRNAs work?
The 5' UTR transcribes a hairpin loop structure that blocks the ribosome binding site which binding of the ribosome, thus having no translation. When sRNAs bind, it binds to the 5' UTR and exposes the RBS for translation to occur
What does Xist RNA do?
The RNA coast or pains the entire inactivated chromosome and binds proteins that mediate chromatin inactivation (causing deacetylation of histone tails)
Which type of CRISPR Cas system is the simplest form? What has this allowed for?
Type 2 system with Cas9 is the simplest form, which has allowed it to be adapted for engineering purposes
What occurs in translation initiaiton with riboswitches?
When there is no metabolite bound, the riboswitch allows for regions 2 and 3 to pair with each other and exposes the RBS for translation. When there is metabolite binding, this changes the RNA structure causing sequence 1 and 2 to pair, and 3 and 4 to pair, in order to block the RBS by the formation of a sequestering helix that forms from 3+4. Thus no translation
What occurs in transcriptional termination with riboswitches?
When there is no metabolite, there is no termination, transcription continues. In the presence of a metabolite (i.e. SAM), this causes regions 3 and 4 to pair with each other and 1 and 2 to pair with each other. The pairing between 3 and 4 form a hairpin structure that acts as a transcription terminator
How does repression using sRNAs work?
Without the sRNA, the RBS is exposed and allows for translation. If the sRNA binds, it binds to the RBS thus blocking the ribosome from recognizing this region for translation-> no translation
What is the function of Xist RNA in mammals and what does it code for?
Xist is required for X-chromosome inactivation (in females one of two X chromosomes is inactivation and in males, no inactivation) and doesn't code for protein but it encodes for the only gene transcribed on the inactivated chromosome
What is within the Paz domain of argonaute protein?
after cleavage, guide RNA is within the Paz domain and it looks for complimentary to different RNAs for cleavage
What is argonaute protein?
aka, slicer that is a multidomain protein
What is a riboswitch?
an RNA structure, usually in a noncoding leader of an mRNA, that controls gene expression upon binding of a small molecule, which is usually a metabolite associated with enzymes encoding in the operon
Where can miRNAs be encoded in stem loops?
can be in either or both sides of the stem loop
What can a single riboswitch control/
can control expression of many genes in an operon, or the same riboswitch can be found associated with different genes that respond to the same ligand
What genes do all subtypes of CRISPR -Cas Systems include?
cas1 and cas2 genes are included, which are responsible for spacer acquisition
What occurs when the riboswitch is bound to the metabolite?
causes a change in the RNA structure of the riboswitch that alters expression of the mRNA
Where is CRISPR-Cas found?
common in eubacteria and nearly universal in archaebacteria
How is CRISP-Cas used to inactivate a gene?
crRNP introduced into cell by microinjection or in vivo expression of components. crRNP recognizes a 30bp by complementarity to tracrRNA and 3bp by protein recognition of PAM. This causes a DSB and repair of DSB using NHEJ usually results in mutations
What are miRNAs?
dsRNAs that are encoded in the nucelus and feed into the RNAi pathway
Function of IncRNAs?
functions are largely unknown, but some clearly have regulatory roles
What do primary miRNA transcripts fold into?
imperfect stem-loops
What is the RITS mechanism important for?
in inhibiting transposon activity
What are some species that need amplificaiton and why?
in some species (e.g. fly and worm), there is an amplification step that increases the effect, perpetuates interference over time and also spreads interference to other cells
what other process can ligand binding mediate?
ligand binding can cause transcriptional termination or anitermination
Where are IncRNAs produced?
long non-coding RNAs (kb in length) stable in cells and can be present in the nucleus or cytoplasm
Where are miRNAs located? (3)
miRNAs are encoded within coding sequences, within introns and UTRs and within "dummy transcriptions) that produce only miRNA to be processed in the RNAi pathway
How many genes are miRNAs thought to target and where?
miRNAs are thought to target roughly half of human genes, usually sequences in the 3' UTR
What is the function of the dicer?
processes dsRNA into siRNAs
How does mRNA degradation work?
sRNAs that are long and antisense can bind to complimentary mRNA and undergo degradation as RNase 3 (dsRNA-specific RNase) recognizes this double stranded RNA region and degrades the dsRNA region bound together
What regulatory pathways are miRNAs a part of?
seemingly all pathways, including in development and disease
What occurs when DNA from a phage is incorporated into a CRISPR array?
the bacterium becomes immune to phages containing that spacer sequence and DNAs containing the spacer sequence are cleave within the cell (foreign DNA)
How are the genes that regulated SAM related to metabolite binding?
the genes that are being regulated by SAM have a feeback mechanism that controls SAM production. If there is increased amounts of Sam then less metabolite would be produced by turning off transcription of the gene and decrease amounts of sam would turn on translation to produce the protein
what is the protospacer
the sequence in the phage that gets integrated into the array
What do sRNAs code for?
they don't code for protein but code for stable RNAs required for regulation