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Molecular Biology > Antisense, siRNA and miRNA > Flashcards

Antisense, siRNA and miRNA Flashcards

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1
Q

How did purple petunias come about?

A

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

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2
Q

Make sense of the results of purple petunia experiment

A

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

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3
Q

What is co-suppression ?

A

Purple petunia experiment- called co-suppression because the experiment suppressed the expression of both the endogenous gene and trans gene

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4
Q

How could extra copies of the gene lead to do-suppression?

A

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

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5
Q

What is the functioning of injecting dsRNA?

A

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

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6
Q

Give the functions microRNAs

A
  • Metabolism
  • Cell cycle
  • development
  • Apoptosis
  • Oxidative stress
  • T-cell activation
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7
Q

Illustrate the discovery of microRNAs

A

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)

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8
Q

Which groups does RNAi occur in ?

A
  • Protozoa
  • Plants (~600 miRNA)
  • Worms. (100-200 miRNA)
  • birds
  • fungi
  • insects (~200 miRNA)
  • fish
  • Mammals (~1500miRNA)
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9
Q

How much of human RNAS

s are a target for miRNA?

A

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

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10
Q

Explain RNAi function in cell regulation

A

Eukaryotes process several hundred different small RNAs

  • embryonic develop
  • cell proliferation
  • cell death
  • fat metabolism
  • cell differentiation
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11
Q

What is the function of RNAi in human disease?

A
  • chronic lymphocytic leukemia
  • colonic adenocarcinoma
  • Burkitt’s lymphoma
  • viral infections
  • bipolar disorder and schizophrenia
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12
Q

What are the naturally-forming small RNAs?

A

miRNA-microRNA

siRNA- short interfering RNA

rasiRNAs-repeat Associated RNA

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13
Q

What is miRNA?

A
  • derived from specific ds-pre-miRNA species
  • regulates expression by repressing mRNA translation
  • mostly endogenous (from the genome)
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14
Q

What is siRNA?

A

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 )
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15
Q

What are rasiRNAs?

A

Repeat associated RNA

-regulates expression by forming heterochromatin

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16
Q

What are artificial miRNAs used for?

A

To study gene function

17
Q

What is the origin of miRNA?

A

From our genome

  • about 60% of miRNAs are expressed independently,
  • 15% of miRNAs are expressed in clusters
  • 25% are in introns
  • MiRNA= MicroRNA
18
Q

What is the origin of siRNA?

A
  • 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
19
Q

What happens to Long Pri-miRNAs?

A

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
20
Q

How does dicer process long dsRNA into siRNA?

A
  • 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
21
Q

Discuss miRNA biogenesis and function

A

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
22
Q

What is one of the first examples of miRNA regulation ?

A

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

23
Q

Recap the mechanism of mRNA degradation by siRNA

A

-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

24
Q

What are the RNA-induced silencing complex (RISC)?

A

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
25
Q

What are the functional domains of the Dicer protein?

A

DEAD box helicase domain= ATP dependent RNA unwinding capacity

PAZ= RNA binding domain

RNaseIII= introduces breaks in dsRNA

NLS= nuclear localization signal

DRBD= dsRNA interaction domain

26
Q

What is the function of rasiRNA?

A

Small RNAs can form a complex with chromatin remodeling enzymes (e.g. DNA methyltransferase or histone deacetylase ) to induce the formation of transcriptional repressed chromatin

27
Q

Explain antisense regulation of gene expression

A

The term antisense refers to the use of a nucleic acid that is complementary to the coding

  • When nucleic acids that are antisense In nature are introduced into cells, they can hybridize to the complementary ‘sense’ mRNA
  • Synthetic antisense DNA chains as short as 15- 17 nucleotides in length have been used to block specific gene expression by either physically blocking translation of the target mRNA or causing its degradation
  • Antisense sequences are designed to specifically inhibit a single gene transcript
  • Antisense therapeutic agents have been made that successfully target HIV, hepatitis B, herpes simplex and papillomavirus infections as well as various cancers
28
Q

Contract Antisense and RNAi

A

Antisense:

  • Single stranded DNA oligonucleotides of variable
  • Doesn’t depend on specific celllulat machinery
  • Not very efficient

RNAi:

  • Large dsRNAs or small (21-35 bp) siRNAs
  • Depends on presence of particular cellular machinery (DICER, RISC)
  • Efficiency depends on availability of cellular machinery in vivo
29
Q

What is Triggering RNAi?

A

RNAi can be triggered

  • Endogenous miRNA
    • regulated miRNA gene expression to control development
  • Exogenous constructs
    • Experimentally delivered, artificial long dsRNA or processed siRNA
    • Promoter driven expression of short-hairpin RNA(shRNA)

-RNA viruses
Some RNA viruses have dsRNA intermediates during life-cycle May encode for miRNAs

30
Q

Describe the normal Bcl-2 activity

A

Bcl-2 is an anti-apoptotic protein, allows cells to live longer

Increase Bcl-2 prevents caspase (protein involved in apoptosis )

This causes cell proliferation to occur

31
Q

What happens when dsRNAs are directed against transcripts of Bcl-2 ?

A

This occurred in two studies:

  1. Human Melanoma MB001 cell culture
  2. Human breast carcinoma MCF-7 cell culture

Both studies resulted in an increase of apoptotic activity (cell death)

32
Q

What is the role of microRNA in normal heart development or disease?

A

Studies conditionally inhibiting miRNA maturation in the murine heart, and has revealed that:
-miRNAs play an essential role in heart development

miRNA expression profiling studies demonstrate that expression levels of specific miRNAs change in diseased human hearts, pointing to their development in cardiomyopathies

33
Q

Describe MicroRNA Dysregulation and cancer

A

Some miRNAs in the genome are amplified or down-regulated in cancer tumors.

These miRNAs are typically found to be involved in regulating cell proliferation

Example:
MicroRNAs: -15a, -16, and 21 are frequently found to be down-regulated in cancer cells. These microRNAs are responsible to arresting cells proliferation (miR-15a and -16) as well as inducing apoptosis (miR-21)

So, if the mRNA is not around to inhibit apoptotic protein synthesis, cell cycle will occur unchecked and promote tumor growth

34
Q

MicroRNAs and RNAi have an extremely…

A

Promising future in both disease treatment and and disease prognosis

35
Q

How can microRNA expression profiles classify human cancers?

A
  • 334 human tumor samples
  • Analyzed array of 217 mammalian miRNA
  • miRNA demonstrated distinct profiles in various types of cancer
  • miRNA profiles better discriminated between tumor tissue of origin, compared with mRNA profiles
  • miRNA ‘signature’ was able to correctly classify 7/12 “carcinoma of unknown primary” where morphology/routine pathology was unrevealing (but the tissue source was known)

Molecular Biology (24 decks)

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