CMMB 411: Topic 12 - Regulatory RNAs

useful in biotechnology

- RNAi makes it easy to experimentally disrupt gene expression in vivo - the effect is a "knock-down" rather than a "knock-out" so it is amenable for studying essential genes; hard to generate mutant - good for testing gain-of-function (GOF) mutations - an engineered short hairpin RNA (shRNA) is expressed in vivo corresponding to the sequence of a desired gene

amplification by RNA dependent RNA polymerases (RdRP)

- RdRP generates dsRNA after recruitment to mRNA by the original siRNA; these dsRNA feed into the system to produce more siRNAs. - more dsRNA = more processing by dicer to create more siRNAs - siRNA-RISC complex can recruit an enzyme (RdRP) to the target RNA, and the siRNA (guide RNA) acts as a primer for RdRP to transform the target into dsRNA. **** produced by dicer, small mRNAs recognized by RISC; one RISC could target many mRNAs - generate more siRNAs, effectively amplifies signal - RdRP found in plants, worms and fission yeast, not in mammalian cells

gene inactivation using CRISPR-Cas9 technology

- cas9-RNP introduced into cell by microinjection or in vivo expression of components - the cas9-RNP binds to DNA at a site complementary to gRNA and adjacent to a PAM sequence, which is essential for cas9's DNA cleavage activity - cells typically repair double-strand breaks (ds breaks) via the NHEJ pathway, which can introduce indwells, often leading to gene knockout due to frameshift mutations *** this method has proven highly successfully for use in eukaryotic genomes - works widely across species (even in bacteria) - technology is very important for genetic studies because it is relatively easy to make mutations at any desired location in a genome

gene silencing

- cleavage of the target mRNA (siRNA) - translation inhibition (miRNA) - transcriptional gene silencing by chromatin modification

overview of siRNAs and miRNAs (part 2) - RISC represses homologous target gene expression in three ways

- directed by siRNAs or miRNA, RISC represses homologous target gene expression in three ways degradation (cut/cleave target mRNA) - attacks and digest mRNAs that has homology - via the "slicer", the argonaute protein (performs initial mRNA cleavage) - usually happens when match between guide RNA and target mRNA is perfect (as in no base-pairing mismatches) translation inhibition - interferes with mRNA translation - usually happens when match between guide RNA and target mRNA is imperfect (as in multiple base-pairing mismatches) chromatin remodeling - directs chromatin modifying enzymes to genes with complementary sequences - happens in nucleus

transcriptional gene silencing by chromatin modification

- in budding yeast S. cerevisiae (lots of centromere repeats), telomeric regions are silenced through histone modifications - centromeric silencing in S. pombe involves the RNAi pathway - centromeric repeats are transcribed from both strands by RNA pol II to form dsRNA, which is then processed into siRNAs by the RNAi machinery (dsRNA => dicer => siRNA) - siRNAs guide the RITS complex to the nascent transcripts tethered to RNA pol II - RITS complex subsequently recruits chromatin-modifying factors, such as Swi6 and Clr4 (constitutive heterochromatin association), which establish a repressive chromatin state by methylating histone H3 at lysine 9 (H3K9me)

translation inhibition (miRNA)

- mechanism is not completely understood - the "seed region" (2-8 nucleotides at the 5' end of the miRNA) is crucial for target recognition - translation is inhibited and mRNA also decays (unsure how RISC subjects mRNA to decay) - miRNA may result in the target mRNA sequestered in processing bodies (P-bodies), where translation is repressed, in the cytoplasm - miRNAs are thought to target roughly half of human genes, usually through sequences in 3'-UTRs - miRNAs are involved in regulation of seemingly all pathways, including in development and disease - regulatory effects can be subtle (e.g. 2x) or massive (e.g. >1000x)

some argonaute proteins have "slicer" activities

- multidomain protein: PAZ domain and RNase H-like domain - PAZ domain recognizes the 3' end of gRNA (siRNA or miRNA) - gRNA base pairs with target RNA - RNase H-like domain (active site) cleaves target RNAs near the middle of the gRNA-target RNA duplex (siRNA), a.k.a. paired region b/w small RNA and mRNA - some argonaute proteins do not have active RNase activity are involved in translation repression (miRNA)

recognition and cleavage of pri-miRNA by drosha/DGCR8 (microprocessor complex)

- pre-miRNA production occurs in nucleus - drosha complex recognize pri-miRNA based on the distinct structure - drosha cleaves 11 bp away from the dsRNA-ssRNA junction - pre-miRNA has 2 nt overhanging on its 3' end, which is important for dicer recognition - pre-miRNA is exported into cytoplasm for dicer processing

ways of RNA regulating gene expression (prokaryotic examples)

- thermosensor for virulence gene expression - small metabolite regulate translation via riboswitch - activation and repression of translation by sRNA

overview of the CRISPR-Cas system

1) after phage infection, surviving cells contain a spacer sequence derived from the phage, incorporated at the beginning of the CRISPR array 2) the CRISPR locus is transcribed and processed into short crRNAs, with each crRNA associating with proteins to form a crRNP complex 3) the crRNP complex surveils DNAs in the cell, and cleaves any DNAs that are complementary to the spacer sequence in the crRNA

how to engineer a cut site

1) find a PAM sequence in the desired target DNA (NGG) - N = any nucleotide 2) select the 20 bp of adjacent sequence as the spacer sequence (5' upstream of PAM sequence) 3) make an RNA molecule containing the spacer sequence (green) fused to the tracrRNA stem-loop (red) 4) deliver the RNA and cas9 protein into a cell (inside it) EITHER: - combine RNA and cas9 in vitro, microinject into nucleus OR: - make a plasmid construct expressing both cas9 and guide RNA and introduce the plasmid into cells (transcribe and translate Cas9) - in this way, an enzyme can be engineered to cleave DNA at any sequence in a mammalian genome (allowing for the PAM sequence) - works for essentially ANY sequence that has PAM site because it is based on base pairing

four steps of CRISPR-Cas function

1) spacer acquisition 2) crRNA processing 3) crRNP assembly and surveillance 4) target degradation

publications on CRISPR

2012 CRISPR-Cas9 is RNA-guided DNA endonuclease jan. 2013 Cas9-RNA mediates site-specific genome engineering in human cells, other eukaryotes, defense from viruses after 2013, the number of publications peaked all the way to present day (about ~1000 publications per year) CRISPR could be used for gene editing

type II-A system from S. pyogenes (a cas9 system)

A) genomic CRISPR locus - maturation and interference aided by transactivity RNA (tracrRNA) - adaptation involves cas operon, encodes cas9 genes B) tracrRNA:crRNA co-maturation and cas9 co-complex formation the natural pathway of antiviral defense involves association of cas9 with the anti repeat-repeat RNA (tracrRNA:crRNA) duplexes, RNA co-processing by ribonuclease III, further trimming, R-loop formation, and target DNA cleavage. - processing only needs cas9 - cas9 directs repeats and recruits - RNase III cleave b/w boundary, spacer sequence, and process transactivity RNA (tracrRNA) - consensus sequence that'll be partially complementary C) RNA-guided cleavage of target DNA details of the natural DNA cleavage with the duplex tracrRNA:crRNA - cas9 scans dsDNA, recognizes PAM sequence - 3 NGG sequences scanned sees whether guide RNA will pair with outer sequence - carry out 2 cleavages via 2 domains. 5' of PAM site to create double-stranded breaks

define CRISPR and Cas

CRISPR: clustered regularly interspersed short palindromic repeats Cas: CRISPR-associated protein gene

RITS

RNA induced transcriptional silencing complex

a mutation in the sequence of prokaryotic DNA encoding the ribosome binding site (RBS) may result in which of the following? A. reduced initiation of translation B. reduced transcription of the mRNA for the gene in which the mutation occurs C. paused ribosome during elongation D. paused RNA polymerase during elongation

a mutation in the sequence of prokaryotic DNA encoding the ribosome binding site (RBS) can result in: A. reduced initiation of translation the RBS in prokaryotic DNA is crucial for the initiation of translation. it's the sequence on the mRNA to which ribosome bind to start translating the mRNA into a protein. a mutation in RBS can hinder ribosome's ability to correctly and efficiently bind to mRNA, leading to reduced or inefficient initiation of translation. this can affect the level of protein synthesis from affected gene. the other options do not directly relate to a mutation in the RBS.

pathway for processing and DNA cleavage (E. coli)

a precursor transcript is generated from the CRISPR array the repeat sequences form hairpin structure ("palindromic repeats") transcript is cleaved at the base of the hairpins to generate single units of one repeat + one spacer (but repeat sequence is divided) proteins with DNase activity associate with the crRNA DNAs in the cells that are complementary to the spacer RNA sequence (light green are cleaved)

which of the following is a shared characteristic of miRNAs and siRNAs? A. miRNAs and siRNAs are both processed in the cytoplasm by the protein dicer B. miRNAs and siRNAs are both processed in the nucleus by the protein dicer C. miRNAs and siRNAs are both processed in the nucleus by the protein drosha D. miRNAs and siRNAs are both made from large hairpin precursors

a shared characteristic of miRNAs (microRNAs) and siRNAs (small interfering RNAs) is: A. miRNAs and siRNAs are both processed in the cytoplasm by the protein dicer both miRNAs and siRNAs undergo a processing step involving dicer, an enzyme that cleaves precursor RNA molecules to produce mature miRNA or siRNA. this processing typically occurs in the cytoplasm. dicer recognizes and cuts dsRNA precursors into shorter, typically 21-23 nucleotide long, RNA duplexes, which are characteristic of mature miRNAs and siRNAs. these small RNAs are then incorporated into the RNA-induced silencing complex (RISC) for their roles in gene regulation of RNA interference

there are many subtypes of CRISPR-Cas systems

all subtypes include cas1 and cas2 genes, which are responsible for spacer acquisition genes involved in crRNA processing, crRNP assembly and target degradation are variable across subtypes - lots of variant systems, relatively little conservation of proteins involved proteins in the crRNP complex are especially variable type II system w/ cas9 is the simplest, which has allowed it to be adapted for engineering purposes

precise genome editing with CRISPR-Cas9 via homology-directed repair

along with the cas9-RNP, a synthetic DNA repair template designed to carry the desired mutation and homology arms that flank the target site for precise integration (of sequence you want to introduce) a double-strand break (DSB) is generated in vivo at the desired site homology-directed pair (HDR) introduces the sequence of the repair template into the genomic sequence (HDR = DSBR) - introduction of specific changes around double-strand break site

which of the following could NOT introduce a premature stop codon in mRNA? A. a mutation in the coding sequence within the DNA B. insertion of an incorrect nucleotide by RNA polymerase during transcription C. a mistake during splicing that leads to retention of an intron D. a mutation in the promoter sequence within the DNA

among the options provided, the one that could NOT introduce a premature stop codon in mRNA is: D. A mutation in the promoter sequence within the DNA the promoter sequence is a region of DNA that initiates transcription of particular gene. mutations in the promoter region can affect the rate of efficiency of gene transcription, but they do not alter the coding sequence of the mRNA. therefore, a mutation in the promoter would not directly result in the introduction of a premature stop codon in the mRNA. all the other options could potentially lead to a premature stop codon.

to what does an "aminoacyl-tRNA" refer? A. any uncharged tRNA B. any tRNA covalently attached to an amino acid at the 3' end of the tRNA C. any tRNA covalently attached to an amino acid at the 5' end of the tRNA D. any tRNA with a 5'-CCA-3' sequence at the 3' end of the tRNA

an "aminoacyl-tRNA" refers to: B. any tRNA covalently attached to an amino acid at the 3' end of the tRNA aminoacyl-tRNA is formed in a process known as tRNA charging, where a specific amino acid is covalently attached to the 3' end of a tRNA molecule. the process is catalyzed by an enzyme called aminoacyl-tRNA synthetase, which ensures the correct amino acid is attached to its corresponding tRNA. the attachment of amino acid to the tRNA is a key step in the translation process, enabling tRNA to deliver appropriate amino acid to growing polypeptide chain during protein synthesis.

components of the small RNA (RNAi) machinery

drosha an RNase-III enzyme cleaves primary-miRNA (pri-miRNA) to form precursor-miRNA (pre-mRNA) that can be recognized by dicer dicer an RNase-III enzyme that recognizes and digests the stem-loop structures of pre-miRNA to form miRNAs or longer dsRNA to form siRNAs note: 1. RNase-III enzymes are specific for dsRNA and the resulting dsRNA product has 2 nt. overhanging on the 3' end 2. dicer and drosha recognize and cleave primary miRNA (pri-miRNA) on the basis of the structure rather than its specific sequence RNA-induced silencing complex (RISC) the effector complex that direct gene silencing using the guide siRNA or miRNA for specificity; include a member of Argonaute proteins argonaute protein the effector protein is RISC (complex), interacts with siRNA or miRNA to carry out target mRNA cleavage (siRNA) or translation repression (miRNA)

guide RNA incorporation and RISC maturation

formation of guide strand RNA and RISC activation: - the short double-stranded RNA (miRNA or siRNA) generated by dicer is incorporated into RISC - dsRNA is denatured into a guide strand and a passenger strand - passenger strand is removed from the complex - resulting RISC is called the mature RISC the guide RNA in the mature RISC recognizes and base pairs with the target mRNA. the guide RNA gives the RISC specificity. - bases between 2-9 nt important in recognizing target mRNA in miRNA (seed residues) offers the highest complementarity

which one of the following steps in prokaryotic translation does NOT require hydrolysis of GTP? A. delivery of correct aminoacyl-tRNAs to the ribosomal A site by EF-Tu B. EF-G stimulation of ribosome translocation C. assembly of the 30S initiation complex D. assembly of the 70S initiation complex

in prokaryotic translation, the step that does not require the hydrolysis of GTP (guanosine triphosphate) is: D. assembly of the 70S initiation complex the assembly of 70S initiation complex in prokaryotes, which involves the joining of the 30S and 50S ribosomal subunits to form the functional ribosome, does not typically require GTP hydrolysis. this step is primarily driven by interactions between the ribosomal subunits and other initiation factors, rather than by energy from GTP hydrolysis. the other options DO involve GTP hydrolysis.

as a measure of quality control in translation, which component of the translation machinery is capable of proofreading and editing amino acids? A. the ribosome B. tRNA syntheses (editing pocket) C. tRNAs D. RNA polymerase

in the context of quality control in translation, particularly with respect to proofreading and editing amino acids, the component responsible for this function is: B. tRNA synthetases (editing pocket) aminoacyl-tRNA synthetases are enzymes that play a critical role in protein synthesis. they are responsible for attaching correct amino acid to its corresponding tRNA, a process known as "charging" the tRNA. many of these enzymes have an editing pocket or function that can remove incorrectly added amino acids. this proofreading ability ensures high fidelity in protein synthesis by reducing the chances of misincorporated amino cids, thus maintaining the accuracy of translation.

type II systems: cas9 complexes

in type II systems, cas9 complex, bound to the CRISPR RNA (crRNA) - transactivity crRNA (tracrRNA) duplex, follows a similar mechanism of PAM-dependent recognition of invading DNA. however, unlike the type I system, the PAM is located upstream (at the 5' end) of the protospacer and both target DNA strands are cleaved by cas9-mediated nuclease activity steps 1) first, the PAM sequence is recognized by cas9 (protein-mediated) 2) then, the DNA target gets progressively unwound to form base with the spacer RNA sequence 3) if there is full complementarity, the nuclease is activated and both strands of the DNA target are cut; DNA is degraded

CRISPR-Cas systems

mechanisms of bacterial defense against phages and foreign DNA common in eubacteria and nearly universal in archaebacteria spacers correspond to 20 to 40 bp complementary sequences derived from phages and plasmids when DNA from a phage is incorporated into a CRISPR array, the bacterium becomes immune to phages containing that spacer sequence DNAs containing the spacer sequence and PAM are cleaved within the cell (foreign DNAs)

microRNAs (miRNAs)

miRNAs are encoded as segments of longer transcripts over 1000 miRNAs have been identified in humans (not a complete list) primary-miRNAs - they are encoded in introns and UTRs, and within exons (e.g. "dummy transcripts" that only produce miRNAs/proteins) - similar to ordinary mRNA 5' cap, and 3' A tail it it possible to predict their presence based on the ability to form the distinct 2nd structures candidate target genes can be predicted based on base-pairing

peptide bond formation is catalyzed by ____________ and occurs between the ____________ sites of the ribosome A. ribosomal proteins; A and P sites B. ribosomal RNA (rRNA); A and P sites C. ribosomal proteins; P and E sites D. ribosomal RNA (rRNA); P and E sites

peptide bond formation is catalyzed by: B. ribosomal RNA (rNA) and occurs between the A and P sites of the ribosome correct answer B. ribosomal RNA (rRNA); A and P sites this process is a key part of protein synthesis, where the rRNA in the large subunit of the ribosome catalyzes the formation of a peptide bond between the amino acid attached to the tRNA in the A site and the growing polypeptide chain attached to the tRNA in the P site. this reaction is a central aspect of the ribosome's function as a ribozyme, demonstrating the catalytic capabilities of RNA.

Q: in which of the following pre-mRNA sequence elements can pre-miRNAs be coded? A. exons, introns, and noncoding regions B. exons and introns C. noncoding regions D. introns and noncoding regions

pre-miRNAs (precursors to microRNAs) can be coded in various regions of pre-mRNA sequences. these regions include: A. exons, introns, and noncoding regions this option is the most comprehensive and accurate. pre-miRNAs can be derived from all these areas within the pre-mRNA exons: while less common, some pre-miRNAs can originate from exon regions introns: many miRNAs are known to be encoded within the introns of protein-coding and non-protein-coding transcripts noncoding regions: these regions, which do not code for proteins, can also contain sequences that give rise to pre-miRNAs microRNAs are a versatile and widespread class of small non-coding RNAs that play crucial roles in regulating gene expression, and they can be encoded in various parts of the genome., including within the sequences of other genes.

dicer

processes drosha-processed pre-miRNA or dsRNAs into miRNAs or siRNAs (siRNA production from dsRNAs only needs dicer) dicer acts of any dsRNA, regardless of sequence, and cleaves it 22 nt. from its 3' end

if this is a stretch of amino acid coding sequence (i.e. coding strand 5' to 3') for a gene A, can you fill in the blanks in the pre-miRNA sequence so that expression of this pre-miRNA in the cell will result in knockdown of gene A expression? (select the sequence you choose and fill in the blanks) GTT GGG GGG AGG GGT CGG CAA TTG AAC CGG TGC CTA GAG AAG

select 21 or 22 nt sequence; make sure the gRNA strand (antisense) is going to be complementary with the predicted mRNA sequence passenger strand GGG GGG AGG GGT CGG CAA TTG guide RNA strand CCC CCC TCC CCA GCC GTT AAC (final answer: change the T's into U's)

some miRNAs and their pre-miRNAs

sequences in red are miRNAs 1 sequence will be future guide miRNA in some cases, both "arms" of a stem-loop can generate a functional miRNA in such cases, the second miRNA is shown in blue-- for example, miR-1 (red) and mi-R1* (blue), as well as with miR-34 (red) and miR-34* (blue). - stem section can sometimes produce 2 guide mRNAs the miRNAs shown are all from the worm. lin-4 and let-7 were identified genetically; those called miR were found by bioinformatics

why is siRNA used in the human biotechnology example instead of dsRNA?

siRNA/synthetic mRNA used instead of dsRNA in order to prevent inducing response in humans - use modified bases to make RNAs to eliminate immune response

overview of siRNAs and miRNAs (part 1)

siRNAs or miRNAs are produced from dsRNA or pre-miRNA by enzyme dicer the siRNAs or miRNAs are incorporated into RISC (RNA-induced silencing complex) and denatured to give a guide RNA, which gives RISC specificity - double-stranded RNA is going to be denatured sequence complementary through base-pairing

determine the actual sgRNA targeting sequence

step 1 identify a PAM (NGG) sequence in the DNA sequence you would like to target step 2 determine the 5' start of the actual sgRNA targeting sequencing by counting 20 nucleotides upstream of the PAM sequence step 3 determine the actual sgRNA targeting sequence many potential 5'-NGG-3' sites if you check the top strand from 5' to 3' direction you can also check the bottom strand for 5'-NGG-3' as well, but it does not have any in this case 5' G AAC CGG TGC CTA GAG AAG G

how is the target specificity determined?

the DNA target (in invading phages) is specified by: 3 bp PAM sequence (protospacer adjacent motif) - recognized by the cas9 protein ~20 bp complementarity to the spacer sequence in the RNA - recognized by RNA-DNA pairing

what is the function of the PAZ domains in dicer and argonaute proteins? A. in both proteins, the PAZ domains recognize and bind the 3' end of a double-stranded RNA molecule B. in both proteins, the PAZ domains recognize and bind the 5' end of double-stranded RNA molecule C. in dicer, PAZ has RNase III activity used to cleave a double-stranded pre-miRNA. in argonaute, PAZ has RNase III activity used to cleave target mRNA strand D. in dicer, PAZ recognizes and binds the 3' end of the double-stranded pre-miRNA. in argonaute, PAZ has RNase III activity used to cleave the target mRNA strand

the PAZ domains in dicer and argonaute proteins have a specific function: A. in both proteins, the PAZ domains recognize and bind the 3' end of a double-stranded RNA molecule. - PAZ domain is an RNA-binding domain found in both dicer and argonaute proteins. - in dicer, PAZ domain contributes to the recognition and binding of the 3' overhangs of dsRNA precursors (such as pre-miRNA) - in argonaute, PAZ domain similarly helps in binding to the 3' end of the RNA strand within the RNA-induced silencing complex (RISC) - this domain does not have RNase III activity; such activity in dicer is responsible for cleaving dsRNA into small interfering RNA (siRNA) or miRNA, but the activity is attributed to other domains in dicer, not the PAZ domain. in argonaute, the slicer activity (responsible for cleaving target mRNA) is also associated with other domains, not PAZ domain.

integration of new spacer sequences

the cas1 and cas2 proteins/processor enzymes (and sometimes other cas proteins too) integrate DNA segments from invading phages into the CRISPR array the sequence in foreign DNA is protospacer (native context) - after integration it is a spacer (in the array) protospacer are next to a 3 bp (in case of cas9 system) PAM sequence (protospacer adjacent motif) only sequence next to a PAM sequence can be integrated into the array the PAM sequence is NOT incorporated as part of the spacer

which of the following proteins combine to form the microprocessor complex used to produce active miRNA? A. pasha (or DGCR8) and drosha B. drosha and dicer C. drosha and argonaute D. pasha (or DGCR8) and dicer

the microprocessor complex, which is involved in the production of active miRNA, is formed by the combination of: A. pasha (or DGCR8) and drosha in this complex, drosha is an RNase III enzyme that initiates the processing of primary miRNA transcripts (pri-miRNAs) by cleaving them to release precursor miRNA (pre-miRNA) hairpins. pasha, also known as DGCR8 (digeorge syndrome critical region gene 8), is a double-stranded RNA-binding protein that assists drosha in recognizing and processing pri-miRNAs. the partnership b/w drosha and pasha/DGCR8 is crucial for the accurate and efficient production of miRNAs in the cell.

CRISPR-Cas and genome engineering

the simplicity of the type II CRISPR-cas9 system make it amenable for engineering cas9 is only protein in the crRNP (other types can have a dozen proteins) there are two RNAs: the tracrRNA and the crRNA (the repeat+spacer RNA) in the laboratory, the two RNAs can be fused to make one RNA the engineering crRNP contains 1 protein and 1 RNA molecule (single guide RNA = single RNA molecule)

gene silencing by short RNAs

three types of small regulatory RNAs - small interfering RNAs (siRNAs): derived from dsRNA (artificially produced or in vivo, sometimes by some cells) - microRNAs (miRNAs): derived from precursor RNAs encoded by genes - piwi-interaction RNAs (piRNAs): predominantly produced in germ lines RNA interference (RNAi) is a powerful tool to silence gene expression in many organism - RNAi: silencing of mRNA expression through short RNA molecules (typically ~21 or 22 nucleotides and can vary from ~19 to 25 nucleotides)