20 - Genome Defense Flashcards
Two main types of genome level threaths
1) invasion of the cell by virus (cellular machinery hijacked by the viral genome).
2) a variety of mobile genetic elements (transposone and retroelements) can insert themselves into the host genome. retroviruses and the bacteriophage Mu share both these enteties.
Mobile elements are present in most genome, but the uncontrolled insertion an dmovement could cause inactivation of manygenes and disintegration from the host genome.
Targeting of the mobile elements with antisense-RNA could prevent them from moving.
transposon = a mobile genetic element that can excise tiself and reintegrate elsewhere in the genome.
retroelements = mobile genetic elements that are transcribed into RNA first before it is converted bact to DNA and inserted elsewhere on the genome.
antisense-RNA = an RNA molecule that is comlpementary to mRNA
noncoding RNA = any RNA molecule that is not translated to give protein.
name some strategies for defending the genome against rouge genetic elements
antisense RNA molecules, enzymatic cleavage of the target nucleic acid, and/or a combination of the two
euk cells also use interferons and antiviral peptides
Antisense RNAs
typically small (<150 nt)
noncoding
complementary to their target sequence. might target mobile genetic elemnts, inherent genes, or a pathogen genome.
play part in controlling gene expression, thus preventing foreign elements being trc/trn to malicious proteins.
restriction endonucleases
cleave DNA at specific internal sequences.
rec short, palindromic repeats
introduce breaks in the sugar-phosphate backbone of the nucleic acid.
RNAi
RNA interference
response that is triggered by the presence of dsRNA and results in the degradation of mRNA or other RNA transcripts homolgous to the inducing dsRNA
sequence specific
RNAi is triggered by dsRNA that is fully base paired and at leasst 21-23 bp in length. Longer dsRNA molecules are fragmented by dicer so the fragment length is 21-23 bp. These RNA molecules are known as siRNA (small interfering RNA), and are recognized bby proteins of RISC (RNA-induced solencing complex).
RISC separates the two strands of siRNA and looks for complementary sequences in the cytosol. If match, the “Slicer”/”Argonaut (AGO) family mmember will degrade the complementary mRNA to prevent the production of viral proteins.
RNAi-related mechaniss can also silence trc or targeted gnes by promoting DNA methylation and altering chromatin structure.
RNAi is not found in prokaryotes, only eukaryotes. However, bacteria do possess ribonuclease III, an enzyme homologous to DIcer which rapidly degrades dsRNA molecules as short as 12bp. Also, bacteria have CRISPR.
Sources of dsRNA that trigger RNAi
RNAi most likely developed as a defense mechanism against viruses, transposons, and transgenes.
for most RNA viruses, the genome will pass through as a dsRNA molecule upon infection (true for ssRNA as well); thus dsRNA is a sign of infection, and triggers an antiviral response.
Dicer enzymes cut the dsRNA into siRNAs. The siRNAs ar erecognized by RDE-1, which recruits RISC. All mRNAs homologous to the viral genome (siRNA) is removed by RISC
microRNA
miRNA
short, regulatory RNA of euk cells.
shares properties with siRNA
blocks translation of mRNA. Usually by binding to the 3’UTR (sometimes in coding region, but less common).
many targets for miRNAencode TFs, supporting the theory that miRNAs are important in gene regulation (of development)
piwi-interacting RNA
piRNA
Piwi is an abbreviation of P-element Induced WImpy testis in Drosophil
an assortment of small RNA molecules involved in regulation. Repetetive seq have been shown to create a large number of piRNAs. The repetetive seqs are often found in clusters tandem repeats areound the centromere and telomere, and scattered repeats (incl transposons) are found throughout the euk genome. some of these repeats include genes that are trc.
longer than siRNA and miRNA (ca 23-26 bp long)
protect against the spread of repeated DNA seqs, and are especially active in the reproductive cells of higher animals.
Dicers
Dicer = general name for enzymes that generate short pieces of RNA that are ca 21-23 nt in length.
domains:
- 2 RNase III domains that work on each backbone of the double helix to create specific cuts
- PAZ domain binds to RNA ends, preferring a 2 nt overhang on the 3’ end of the RNA
- on the opposite side of the PAZ domain is a dsRNA-binding domain that holds the RNA strands in place until RNase III makes the cuts.
when modified Dicer may also take part in degrading DNA (rather than RNA) during programmed cell death.
the distance between the PAZ and RNase domains dictates the length of the siRNA.
Argonaut family
three families:
1) Piwi (binds to piRNA)
2) Ago (binds to miRNA and siRNA)
3) found in nematodes, identified but not studied.
Ago is the central molecule in RISC, and it unwinds the small RNAs created by Dicer to make a ss template called the guide strand. The other srtand of the siRNA, miRNA, and piRNA is discarded. The guide strand is loaded into the Ago protein, and the enzyme searches the cytoplasm for complementary seq.
Ago proteins have PAZ domains that binds to the 3’ end of RNA, a Mid domain that binds the 5’ end of RNA, and a tract of positively charged AAs in between that attacts the negatively-charged phosphates of the guide RNA.
When this complex finds a complementary mRNA in the cytoplasm, it cuts the middle of the mRNA with its RNase III like domain. The strands are then further degraded by RNases. If the guide strand only partially binds to the target mRNA, the Ago does not cut but instead blocks trn.
These activities occur in the P body in the cytoplasm (filled with mRNA degradation enzymes).
Amplification and spread of RNAi
RNAi is very potent
<50 molecules of siRNA can silence target RNA preset in thousands of copies per cell. This is enabled by RNA-dependent RNA polymerase (RdRP) which makes more siRNA copies.
Cutting of the target mRNA by Ago gives two aberrant and unstable RNA molecules, one capped but without poly(A)-tail, ant the other with tail but no cap.
It seems like one of these two are used as a template by RdRP to generate dsRNA, which will act like a substrate for Dicer, generating more siRNA (secondary siRNA), and thus siRNA is amplified.
The RNAi effect can also spread from cell to cell
Delivery of siRNA and applications of RNAi
The use of RNAi can be used to study gene function, it does not require mutants with defect versions to be made. very useful in euks.
Experimentally, RNAi may be induced by providing long molecules of dsRNA that are cut into siRNA by the Dicer enzyme. ss antisense RNA against cellular genes can also trigger RNAi by base pairing with the corresponding plusstrand, generating dsRNA in the cell. Short dsRNAs can also be administered directly and act as siRNA.
Usually more convinient for the dsRNA to be made in vivo. 3 main variations:
1) a single DNA semnent trc from a single promoter that generates a stem and loop structure. In this case the + and - strands are in tandem but separated by a short strentch of DNA that remains unpaired and forms the loop.
2) A DNA segment flanked by two opposing promoters. Consequently, one promoter trc the template and the other trc the sense strand from the same dsDNA segment
3) Two DNA segments, one being the inverse of the other and both having separate promoters. Consequently, one promoter trc the plus strand from the sense version of the DNA, the other trc the minus strand from the other inverted antisense DNA segment.
siRNA in theraputic purposes
cant simply be injected, as they are unstable in physiological conditions and not really taken up by cells. Also high levels can activate an immune response.
Has been used as therapy for cancer, acute kidney diorders, eye diseases, and other pathoglogies related to cell cycle control, angiogenesis, and apoptosis.
Current delivery for medical purposes (4):
1) Chemical modifications can increase the stability (changes to the 2’ position of ribose by addition of 2’-O-methyl or 2’-deoxy-2’-fluoro to increase stability and resist nuclease cleavage)
2) Lipid-based delivery systems include stable nucleic acid-lipid particles (SNALPs). These contain siRNA in a lipid bilayer that is stable in blood and easily taken up by cells. SNALPs have been used successfully in vivo during clinical trials of siRNA therapies for some cancer treatment, HepB ++. Exosomes (extracellular vesicles from host own DNA, occur naturally in blood). Can carry the siRNA over long distances and deliver to specific cell types.
3) Polymer-based systems include water-soluble polymers and polymer nanoparticles. Typically solid biodegradable.
4) Conjugation of siRNA directly to delivery materials. Used for targeting certain cancers.
CRISPR (general)
Clustered Regularly Interspaced Short Palindromic Repeats
Consists of a memory bank of hostile foreign genetic seq + a mechanism for identification and destruction of incoming foreign DNA/RNA (RNAi can only protect against RNA). 90% archaea + 70% bacteria.
some are incomplete/defective.
CRISPR parts
CRISPR memory bank (CRISPR array) found on bacterial chromocome, consists of an array of foreign DNA segments (spacers/memories) alternating with identical repeated seq (can be palindromic, depends on the system).
Genes upstream this array encode the CRISPR proteins (Cas (CRISPR associated proteins) proteins) These have 2 roles:
1) some obtain and store seg of foreign seq (spacer aquisition/adaptation).
2) use the stored seq info to rec and degrade introduc nucleic acids (expression and interferance, respectively).
