Antisense, siRNA and miRNA Flashcards
(35 cards)
How did purple petunias come about?
Attempted to overexpression chalone synthase (anthrocyanin pigment gene) in petunias to make “Deeper purple”
Jorgensen research groups introduced a pigment producing trans gene into petunias with the hope f deepening the color of the flowers
Surprisingly, the flowers were variegated or sometimes completely white
Make sense of the results of purple petunia experiment
Researchers measured the amount of mRNA for the anthrocyanin pigment gene
The mRNA in the transgenic plant is greatly reduced thus the protein is greatly reduced
What is co-suppression ?
Purple petunia experiment- called co-suppression because the experiment suppressed the expression of both the endogenous gene and trans gene
How could extra copies of the gene lead to do-suppression?
The worm held the answer
In 1998, Fire and Mello injected single strand and double-stranded RNA into C. Elegans to generate mutant phenotypes
- In 2006, Andrew fire and Craig C. Mello shared the Nobel Prize in Physiology or Medicine for their work called RNA interference in nematode worm C. elegans
- “Arguably the most important advance in biology in decades (Nature magazine)
Injection of small amounts of dsRNA into C. Elegans produced strong knockdown of gene function (loss of protein)
Max-3 (muscle excess protein-3) is a translational regulator in C. Elegans that participates in maintaining the germline totipotency Staining for Mex-3 RNA
What is the functioning of injecting dsRNA?
Injection in gonads of dsRNA for max-3 (abundant RNA gave much more efficient inhibition in embryos than antisense RNA
- dsRNA had to include exons; introns and promoter didn’t work
- Effect was incredibly potent and even spread to other cells within the worm
Termed ‘RNA interference’ incredibly useful as a tool for molecular biology
Give the functions microRNAs
- Metabolism
- Cell cycle
- development
- Apoptosis
- Oxidative stress
- T-cell activation
Illustrate the discovery of microRNAs
1993- Lin-4: antisenses RNA C. Elegans development
2002- miRNAs discovered in human, mouse, and Drosophilia
2004- miRNAs implicated in leukemia
2005- miRNAs identified in viral genomics
2008- miRNAs proposed to be involved MRD (Bcell lymphomas)
Which groups does RNAi occur in ?
- Protozoa
- Plants (~600 miRNA)
- Worms. (100-200 miRNA)
- birds
- fungi
- insects (~200 miRNA)
- fish
- Mammals (~1500miRNA)
How much of human RNAS
s are a target for miRNA?
1/3 of human. RNAs may be targets of miRNA
A complex set of biochemical mechanisms has been conserved by evolution to facilitate microRNA mediated gene expression control
Explain RNAi function in cell regulation
Eukaryotes process several hundred different small RNAs
- embryonic develop
- cell proliferation
- cell death
- fat metabolism
- cell differentiation
What is the function of RNAi in human disease?
- chronic lymphocytic leukemia
- colonic adenocarcinoma
- Burkitt’s lymphoma
- viral infections
- bipolar disorder and schizophrenia
What are the naturally-forming small RNAs?
miRNA-microRNA
siRNA- short interfering RNA
rasiRNAs-repeat Associated RNA
What is miRNA?
- derived from specific ds-pre-miRNA species
- regulates expression by repressing mRNA translation
- mostly endogenous (from the genome)
What is siRNA?
Short interfering RNA
-derived from long dsRNAs and random processing
- regulates expression by mRNA degradation
- often exogenous (from outside the cell, I.e. virus or injected )
What are rasiRNAs?
Repeat associated RNA
-regulates expression by forming heterochromatin
What are artificial miRNAs used for?
To study gene function
What is the origin of miRNA?
From our genome
- about 60% of miRNAs are expressed independently,
- 15% of miRNAs are expressed in clusters
- 25% are in introns
- MiRNA= MicroRNA
What is the origin of siRNA?
- double stranded RNA molecules
- Mainly introduced to cell from outside
- Can be made by a scientist and transferred into cells
- Can be made by a virus that infects the cell
- Some have recently been found to originate from our genomic DNA
What happens to Long Pri-miRNAs?
Long Pri-mRNAs are processed to pre miRNAs hairpin structures (~70 nt) by Drosha)
- Pre-miRNA transported to cytoplasm by Exporting 5
- Dicer further processes them to single stranded RNA and initiates the formation of the RNA-induced silencing complex (RISC)
- RISC binds imperfectly to the 3’ untranslated region of TARGET mRNA
- Inhibition of the ribosome ability to translate protein from mRNA transcript
How does dicer process long dsRNA into siRNA?
- Dicer also processes long dsRNA into siRNA (small interfering RNA)
- siRNA has perfect complementary pity with the 3’ untranslated region of target mRNA
- Perfect complementary leads to endonuclease activity and target mRNA is cleaved, no protein is translated from the destroyed mRNA
Discuss miRNA biogenesis and function
1/3 to 2/3 of all mRNAs may be regulated by mRNA
miRNA genes are found in intergonic regions in defined transcription units on both sense and antisense direction
- miRNA generally transcribed by RNApol-II forming primary miRNA (pri-mRNA) transcripts which have 5’ cap and poly-A tail
- pri-miRNA has stem loop regions that are cropped to about 70 NT by Drosha forming 2 NT 3’ overhang called pre-miRNA
- Pre-mi-RNA exported to cytoplasm by Exporting 5
- The pre-miRNA is processed by Dicer into the ~20 nt mature miRNA which will bind target sequences in the 3’ UTR of mRNA and block translation
- RISC + mRNA known as miRISC
What is one of the first examples of miRNA regulation ?
MicroRNAs regulate gene expression by binding to partially complimentary sites in the 3’ UTR of a mRNA and subsequent inhibition their translation by interfering with ribosome
In C. Elegans, Lin 4 encodes a 22 NT nom-coding RNA that binds to 7 conserved sites located in the 3’ UTR of the lin 14 gene
Recap the mechanism of mRNA degradation by siRNA
-Long dsRNA molecules converted to ~20 nt siRNAs by Dicer (RNAse III-type endonuclease)
- This creates RNAs with 2-nt overhangs
- The two strands of siRNA are sense and anti-sense of the mRNA
- Antisense called guide strand-template for sequence specific gene silencing
- Sense strand is called passenger strand
-The guide strand of siRNA will assemble with RISC to form RNA/protein complex (siRISC) which binds target mRNA and cleaves target RNA thus silencing gene expression
What are the RNA-induced silencing complex (RISC)?
Known components include Dicer, Argonaute proteins, various RNA binding (RBP)
Human argonaute proteins, AGO1 & AGO2 contain a PAZ domain (binds small RNA helicies) and PIWI domain which recognizes dsRNA
- Dicer may have secondary helicase activity
- AGO1 is required for miRNA directed RNAi type inhibition of translation
- AGO2 will cleave target mRNA ~10 NT from the 5’ end of the siRNA guide strand
