Ch. 5 Based Technologies Flashcards
Which of the following statements about antisense RNA is true?
All of the above statements are true
Which biological function is not controlled by antisense RNA?
Replication of prokaryotic genomic DNA
Which of the following is a modification of antisense oligonucleotide structure to increase intracellular stability?
All of the above
How can antisense RNA be expressed within a cell?
The target gene can be cloned inversely into a vector and under the control of an inducible promotor
Which of the following terms describes when gene regulation occurs by short is dsRNA molecules triggering an enzymatic reaction that degrades the mRNA of a target gene?
All of the above
- post-transcriptional gene silencing
- quelling
- co-suppression
- RNA interference
Which statements about RNAi is not correct?
Non-specific interactions between the antisense siRNA and mRNA often cause mRNAs to be degraded that should not have been
Which of the following is NOT a method for delivering dsRNA or RNAi into Drospholia or C. elegans?
Injection of dsRNA into eggs
How can RNAi be triggered in mammalian cells?
All of the above
- transfection of siRNA
- chemically synthesized siRNA
- degradation of target mRNA through shRNA creation
- modification is an existing shRNA to recognize a different mRNA
What information has been obtained through the creation of RNAi libraries?
The function of unknown proteins by degrading all of the mRNA for that protein
What is a ribozyme?
An RNA molecule that binds to specific targets and catalyzes reactions
Which of the following is a large ribozyme?
Twort ribozyme
What process is used to identify possible ribozyme substrates?
DNA SELEX
What property must a ribozyme possess in order to be used in clinical medicine?
All of the above
- stability and resistance to degradation
- no deleterious side effects to the host
- expression within a diseased cell only
- be able to be delivered to the correct location
What is a riboswitch?
An mRNA sequence that binds directly to an effector molecule to control the translation of the mRNA into protein
Which of the following is NOT an example of an effector molecule for riboswitches?
Antisense RNAs
Which RNA is incorrectly paired with its function?
piRNA - RNA processing
Which of the following helps replicate telomeres?
TERC
In drosophila, two non-coding RNAs called roX1 and roX2 are used to…
Idk
What is the role of the alpha antisense form in PTEN expression?
Idk
What role do piRNAs play?
Idk
snRNA -
RNA processing
circRNA -
Transcriptional regulation
Xist -
Chromosomal structure
lncRNA -
Transcriptional regulation
Roles of non coding RNA
- take part in translation; tRNA and rRNA
- introns splicing: snRNA, snoRNA, gRNA
- catalyze enzyme reaction; ribozyme
- genomic integrity
- protection
- regulate gene expression
___ is an enzyme that used an RNA component (TERC) to regenerate the ends that are not created during replication, maintaining the chromosome structure.
Telomerase
___ ___ ___ contains the active site for DNA synthesis.
Telomerase reverse transcriptase (TERT)
Gene dosage compensation
In Drosophila, two noncoding RNAs called roX1 and roX2 complex with five different proteins to form ___ ___.
MSL complex
Gene dosage compensation
Which noncoding RNA coats the inactive X chromosome?
Xist
Gene dosage compensation
The Xist gene of the active X chromosome is inactivated by ___.
Methylation
Piwi-interacting RNAs
The ___ are encoded in genome in large clusters or within introns of other genes.
piRNAs
Piwi-interacting RNAs
They are complementary in sequence to ___ ___.
Endogenous transposons
Piwi-interacting RNAs
The __ __ binds and cleave any complementary RNA produced by transposon.
PIWI complex
Antisense RNA
Transcription uses the antisense strand as a template, resulting in an __.
mRNA
___ ___ is synthesized using the sense strand as template.
Antisense RNA
___ ___ ___ are complementary to a target mRNA, blocking either protein translation or splicing of introns.
Antisense RNA sequences
RNA modulates transcription: antisense RNA
Antisense DNA targets mRNA for ___.
Degradation
RNA modulates transcription: antisense RNA
The ___ ___ encodes three noncoding RNAs: PTENpg1, asRNA alpha, asRNA beta.
PTEN pseudogenes
RNA modulates transcription: antisense RNA
The ____ interact with DNMT3A and EZH2 to condense the histones around the PTEN gene.
asRNA alpha
RNA modulates transcription: antisense RNA
The ___ and ___ prevent the miRNAs from promoting the degradation of normal PTEN mRNA.
PTENpg1 and asRNA alpha
RNA modulates transcription: antisense RNA
___ ___ are synthesized chemically and injected into a cell to block mRNA translation.
Antisense oligonucleotides
RNA modulates transcription: antisense RNA
___ ___ are cloned in inverted orientation so that the sense strand is transcribed to yield antisense RNA.
Antisense genes
The ___ ___ anneals to the normal mRNA, preventing its expression.
Antisense RNA
Antisense RNA -
Backwards compliment of the normal RNA for a target gene
Antisense RNA is designed to..
Bind the target mRNA and prevent translation
Antisense oligonucleotides -
Short sequences designed to hybridize with mRNAs and prevent translation
___ ___ of cellular proteins can be used to determine if the protein production is reduced.
Proteomic analysis
The oligonucleotides are susceptible to degradation by ___ ___.
Intracellular nuclease
First generation therapeutic agents
- modification includes adding sulfur to the phosphodiester backbone
- the RNA-DNA duplex activates the Rnase H which cleaves hybrid molecules
Second generation antisense therapeutic agents
- typically contain alkyl modifications at the 2’ position of the ribose
- less toxic and more specific than phosphorothioate-modified molecules
Third generation antisense oligonucleotides
- contain a variety of modifications within the ribose ring, and/or the phosphate backbone, as well as being less toxic
- some modifications are made to both enhance stability and facilitate the binding to the target site
RNA modulates transcription: antisense RNA
Their use is restricted to __ __ or __ __ __ __ since they are resistant to Rnase H.
Splice sites or block ribosome binding sites
RNA modulates transcription: antisense RNA
___ ___ with Rnase H-sensitive cores are employed.
Chimeric oligonucleotides
RNA modulates transcription: antisense RNA
___ ___ ___ are not easily internalized by cells.
___ is often accomplished using liposomes to facilitate cellular uptake.
Free antisense oligonucleotides
Delivery
Correction of a mutant splice site with an antisense oligonucleotides can be used to treat ___.
B-thalassemia
RNA modulates transcription: RNA interference
- double-stranded RNA, produced by virus or microRNA from genome, triggers RNA interference
- dicer recognizes dsRNA and cuts it into 21-23 nucleotides
- a kinase phosphorylates the 5’ end of each piece
- RISC unwinds the siRNAs and uses one strand to search out complementary mRNA, which later is cleaved by Argonaut
RNA modulates transcription: RNA interference
Amplification of RNAi
- after mRNA is cleaved, RNA-dependent RNA polymerase binds to some of these fragments and synthesized complementary strands
- dicer recognizes these fragments and creates more siRNA
- the RISC complex containing the single-stranded siRNA can also recognize and bind to complementary DNA sequence
- when RISC associates with a repetitive DNA element, various histone-modifying enzymes and silencing complexed are activated
- the regions become heterochromatin, thus silenced
RNA modulates transcription: MicroRNAs
The gene for the miRNA is transcribed into an RNA that folds into a ___ ___.
Stem loop
RNA modulates transcription: MicroRNAs
___ cleaves pri-miRNA to create pre-miRNA.
Drosha
RNA modulates transcription: MicroRNAs
In cytoplasm, ___ cuts the end of pre-miRNA to form a mature miRNA.
Dicer
RNA modulates transcription: MicroRNAs
___ ___ creates the single-stranded template and searches the cytoplasm for any matching sequence.
RISC complex
Amplification of RNAi for studying gene expression
C. elegans can take up dsRNA by…
- ingesting transgenic bacteria expressing dsRNA
- bathing in a solution
- by injection
RNAi for studying mammalian genes
RNAi can be triggered in mammalian cells using ___ ___ ___.
Chemically synthesized siRNA
RNAi for studying mammalian genes
The dsRNA shorter than 30 nucleotides activated the mammalian counterparts of ___ and ___.
Dice; RISC
RNAi for studying mammalian genes
Two ___ at the 3’ overhang makes it more stable.
Uracils
RNAi for studying mammalian genes
___ group can be added to 2’-OH ribose.
Methyl
RNAi for studying mammalian genes
Purified dicer can also be used to produce ___.
siRNA
RNAi for studying mammalian genes
___ can be designed to express different shRNA molecules.
Vectors
RNAi for studying mammalian genes
Two complementary sequences about 20 nucleotides in length separated by a __ __.
Loop region
RNAi for studying mammalian genes
When the vector is transformed into a cell, the shRNA is transcribed and activates ___ ___.
Gene silencing
___ ___ are designed to express dsRNA for each gene in the genome.
RNAi libraries
RNAi libraries are used to..
Identify the role of unknown proteins
Mammalian cells can be screened for defects induced by an RNAi library clone using a ____ or ___.
Live cell microarray
Multi-plate assay
Eukaryotic mRNA splicing
Researchers had expected the complementary of upstream (5’) and downstream (3’).
However, this idea was soon ___.
Discounted
Eukaryotic mRNA splicing
The sequences at 5’ and 3’ are not ___, yet not ___.
Complementary; random
Eukaryotic mRNA splicing
Almost all introns begin with ___ and end with ___.
G:U
A:G
Eukaryotic mRNA splicing
The branch site -
Sequences with introns, 20-50 nucleotides just upstream of 3’, is essential for splicing
Eukaryotic mRNA splicing
Splicing is carried out by a complex of proteins and RNA called the __.
Spliceosome
Alternative mRNA splicing
- often produces two forms of the same protein that are necessary at different stages of development or in different types of cells
___ of the lgM class exist either a membrane bound protein displayed on the cell surface or as a soluble protein secreted into the blood.
Immunoglobulins
___ are mRNA sequences that bind directly to effector molecules to control the expression of the mRNA into protein by attenuation or translational inhibition mechanisms.
Riboswitches
__ __ __ occurs during growth conditions.
Cell wall synthesis
Cell wall synthesis
When the cell is growing, levels of UDP-GIcNAc are low and quickly incorporated into cell wall
If the cell is not growing, the excess UDP-GIcNAc binds to a riboswitch on the glms gene
Activates the self-cleaving ribozyme to degrade mRNA
Small naturally occurring ribozymes
Found in small subviral agents such as viroids and satellite viruses
They have common motifs that catalyze RNA cleavage
Engineering ribozyme for applications
Adding a ribozyme motif such as the hammerhead or hairpin region from the small ribozymes can make antisense oligonucleotides more stable because they are not degraded.
These constructs cut the target mRNA without the use of Rnase H
In vitro selection of ribozymes
Can also generate new ribozymes by mixing random sequences that represent potential ribozymes with a specific substrate
In vitro evolution of ribozymes
Adding a mutagenesis step to the in vitro selection procedure allows the ribozyme to “evolve” into a better enzyme
Combining riboswitches and ribozymes
Ribozymes that only act in presence of effector molecules
Antisense
RNAnase H
RNAi
RISC
Ribozyme
Cuts by itself
