Week 9 - Non-coding RNAs eg siRNAs

human genome actively transcribed into RNA

60 -70%

protein-coding genes

less than 2% of the human genome

non-coding RNA (ncRNA) molecules

Most of the ncRNAs have been widely implicated in regulating cellular processes

- rRNA, tRNA, microRNAs, long non-coding RNAs (lncRNAs), and other regulatory RNAs

Regulate protein synthesis by regulating mRNA

mRNA is degraded before translation begins, the encoded protein will not be produced

Long non-coding RNAs (lncRNAs)

lack an open reading frame and do not code for protein

Exhibit a wide range of secondary and tertiary structures compared to the coding transcriptome (mRNA)

Long noncoding RNAs (lncRNAs) have some features in common with coding RNAs (mRNAs)

can be transcribed by RNA polymerase II or RNA polymerase II

introns

exons

splice variants (alternative splice forms)

transcription is driven by promoter elements

transcription factors- 5'-cap, PolyA tail

tertiary structure of the LncRNAs

lncRNAs are folded after transcription to create tertiary RNA structures and the tertiary structure determines the function of the LncRNAs

Gene silencing

switching off or turning off a protein coding gene that would normally be turned on (expressed)

Transcriptional gene silencing (TGS)

silence genes at the level of transcription, preventing the production of mRNA

How TGS occurs

processes such as DNA methylation, histone modification, etc., which alter chromatin structure to repress transcription

Post-transcriptional gene silencing (PTGS)

suppresses gene expression after transcription has occurred, primarily by degrading mRNA or blocking its translation

RNA interference (RNAi)

prevents the production of the encoded protein

PTGS is often mediated by small RNAs

small interfering RNAs (siRNAs)

microRNAs (miRNAs)

double-stranded RNA serves as the trigger

RNAi mechanism by siRNA recognizes the dsRNAs and works to degrade them

PTGS or RNAi mechanism

• Offers protection from dsRNA viruses

• Regulate gene expression by silencing genes

RNAs expressed from genes are single stranded so...

Double-stranded RNAs are recognized as foreign by the cell, activating an endogenous mechanism that destroys them

Normal mechanism of transcription

antisense DNA strand (3' to 5') is transcribed to produce a sense mRNA strand (5' to 3')

synthesize RNA exclusively in the 5' to 3' direction, requiring a 3' to5' DNA strand as a template

RNA nucleic acid change reminder

Always replace T with U in your RNAs

RNA Polymerases

DNA-dependent enzymes that catalyze transcription; cannot carry out synthesis from the 5' to3' DNA strand

mRNA (sense RNA, 5' to 3') bound to an anti-sense RN

cannot go on to make protein. Thus, the gene from which this mRNA comes is silenced since it cannot express itself by making a protein

RNA-dependent RNA polymerase (RdRp)

synthesize RNA from an RNA template, playing a crucial role in the replication of RNA viruses and certain cellular processes

- present in viruses, plants, fungi, protists, and some animals

antisense RNA

• does not stay base-paired with the template long-term

• RdRP unwinds the antisense RNA during synthesis by strand displacement

• released as a separate single strand

Sense + antisense RNA can anneal

form dsRNA

genetic engineering (cloning technology)

introduce antisense RNAs into human cells that are complementary to mRNA

What is inserted in genetic engineering

provide animal cells with antisense RNA that is complementary to the mRNA

Result of genetic engineering

mRNA will be destroyed, and the protein that should be made from the mRNA will not be made

Gene is silenced

dsRNA is not degraded directly in this form

it is cleaved/cut into small pieces of dsRNAs called small interfering RNA(siRNA) through a cellular pathway

siRNA pathway

long dsRNA is cleaved into ds siRNA

dsRNA, siRNAs made into single strand siRNA; one strand is degraded

other ss siRNA bind to complementary mRNA and the mRNA is cleaved/degraded

siRNA amplification

RdRP synthesizes antisense RNA using target mRNA, forming dsRNA that is processed into secondary siRNA

ENDOGENOUS

Genes already present in organism

Endogenous Sources of long dsRNA

RdRp can use mRNA (5 to 3') to make antisense RNA (3' to 5')

Transposable elements

EXOGENOUS

Adding more copies of already endogenous genes into organism

Exogenous Sources of long dsRNA

Viral infection

Artificial introduction

Viral infection

Many RNA viruses generate dsRNA intermediates during replication, which can activate the host RNAi response

Artificial introduction

Researchers can introduce dsRNA into cells

Synthetic siRNAs

Chemically synthesized siRNAs that mimic naturally processed siRNAs

Short hairpin RNAs (shRNAs)

Engineered RNA molecules that form dsRNA hairpins and are processed into siRNAs by the RNAi machinery

Vector-based expression systems

Plasmids or viral vectors can be engineered to produce dsRNA in cells

natural RNAi mechanism by siRNA is sequence-specific

a piece of RNA (~22) binds to a complementary sequence on the mRNA perfectly, which leads to degradation of the mRNA

RNAi by siRNA is a conserved biological response

RNAi mediated by siRNA have been found in many organisms, including plants, fungi, worms, Drosophila, mice, and humans

siRNA destroys dsRNA, and by doing so

mediates protection to both endogenous parasitic and exogenous pathogenic nucleic acids

regulates the expression of protein-coding genes

Discovery of PTGS mediated by small interfering RNAs (siRNA)

discovered as a naturally occurring pathway accidentally in experiments with TRANSGENES

What is a transgene?

gene introduced into an organism using genetic engineering techniques, either from another organism or as additional copies of an existing gene to enhance the production of its encoded protein

Where must a transgene get into for transcription to occur?

The nucleus of the cell.

transgenesis/recombinant DNA technology

has the potential to change the phenotype of an organism

agrobacterium tumefaciens

used to introduce genes into plant genomes

Transgene induced post transcriptional gene silencing

Transgene X makes antisense RNA X

Endogenous gene X makes mRNA X

RNA X from the transgene X binds to the endogenous mRNA X

mRNA does not make a protein

Experiment that determined TGS

They wanted to make Intensely purple petunia flowers and White petunia flowers

Chalcone synthase

anthrocyanin pigment gene in petunia

enzyme at start of biosynthetic pathway for anthocyanins

More copies of CHS gene

more CHS mRNA --> more CHS enzyme to convert --> more substrate (coumaric acid) into chalcone --> leading to more anthocyanins --> deeper coloration

antisense CHS mRNA will bind to sense CHS mRNA

less CHS enzyme --> less anthocyanins -->white / whitish coloration

Result of experiment

they saw a mix of deeper colored flowers (expected) loss of pigment (unexpected)

the sense construct

Instead of exhibiting more pigment, most petunias displayed reduced color, with some areas completely white

the antisense construct

instead of exhibiting less pigment, most petunias displayed areas of pigment

Northern blots

a technique used to assess gene expression by determining whether a gene is producing mRNA and measuring the quantity of mRNA being synthesized

EGF and TGF genes

exhibit low expression in normal cells but show high expression in cancer cells

small interfering RNA (siRNA)

When there is an excessive amount of mRNA, the cells perceive it as an aberration. As a response, organisms with RdRp gene made the RdRp protein, which uses some of the 5' to 3' mRNA to generate antisense (3' to 5') mRNA

RdRp enzymes

made in response to an overabundance of a specific mRNA (in this e.g.it is CHS mRNA) because the cell view this as abnormal, so it wants to regulate this excessive levels of mRNA.

long double stranded RNA

The sense and antisense from RdRp bind

processed into small RNAs (siRNA) of about 21 to 24 bp

RNA dependent RNA Polymerase (RDRP)

uses the CHS sense mRNA to make antisense mRNA...

BECAUSE THE OVERABUNDANCE OF CHS mRNA IS VIEWED AS ABNORMAL and it wants to regulate the amount of CHS mRNA in the cell

RdRP

using the 5' to 3' transcript as a template to make 3' to 5' antisense transcripts that are complementary to each other

- ubiquitous in plants

- In animals, it has been identified in only a few species

Why is silencing uneven?

1. Variable uptake of RNAi molecules

2. Differences in RNAi machinery

3. Amplification differences (in some organisms)

4. mRNA expression differences

Variable uptake of RNAi molecules

Not all cells receive the same amount of:

• dsRNA

• siRNA

Less RNA → weaker silencing

Differences in RNAi machinery

•Levels of silencing enzymes e.g.,

• Dicer

• Argonaute (RISC)

Cells with more machinery → stronger silencin

Amplification differences (in some organisms)

•Some cells produce more secondary siRNA

Stronger effect

mRNA expression differences

Cells expressing more of the target gene may show:

partial silencing instead of complete knockdown

What uneven silencing looks like experimentally:

• Patchy phenotype

• Partial reduction in gene expression

• Different levels of protein in different cells

Sources of long dsRNA

In plants, RdRP can make antisense RNA from sense RNA. The antisense mRNA will bind to sense RNA.

In humans, if antisense RNA is provided, it can bind to the sense mRNA

Step 1 of siRNA

The long dsRNA is cleaved by the enzyme Dicer (this is an endoribonuclease enzyme, so ribonuclease activity) into 21-24 bp small interfering RNAs (siRNAs)

dicer

enzyme that cleaves and processes double stranded RNA to produce siRNAs or miRNAs that are 21-25 nucleotids in length

Step 2 of siRNA

The double-stranded siRNAs are loaded into RISC

In RISC is ARGONAUTE (Ago), which is an endoribonuclease.

In the RISC complex, the ds siRNA strands unwind to become single stranded and the 5' to 3' strand called the passenger strand is degraded. The 3' to 5' strand, called the GUIDE strand, binds to the AGO protein

RNA-Induced Silencing Complex

uses siRNA as a guide to identify complementary mRNA, then acts as an endonuclease (via Argonaute protein) to cleave and degrade it

Argonaute

Catalytic subunit of the RISC complex of the RNAi machinery. Responsible for the cutting (or "slicing") activity of RISC that destroys target mRNAs.

Step 3 of siRNA

RISC complex with the guide strand bound to AGO is guided to an mRNA (i.e., the target) to which the guide strand then binds complementary to the mRNA (target recognition)

guide strand

21-24 nucleotide strand will bind to a specific region of the mRNA with perfect complementarity.

passenger strand

Strand that is degraded and decomposed after cut by dicer in RNA

Step 4 of siRNA

The AGO, which is an endoribonuclease, cleaves the mRNA.....The cleaved mRNA is then degraded, it cannot be translated to a protein

Strand Selection within RISC

he strand with the less stable (lower GC content) 5′ end is typically selected as the guide strand, while the opposite strand is discarded

Step 1 siRNA amplification by RdRP

RISC binds the target mRNA (Step 3)

- The siRNA inside RISC pairs with the target mRNA.

Step 2 siRNA amplification by RdRP

RdRP uses this mRNA as a template

RNA-dependent RNA polymerase (RdRP) copies the target mRNA.

This creates new double-stranded RNA (dsRNA)

Step 3 siRNA amplification by RdRP

Dicer cuts this new dsRNA again

The new dsRNA is processed into more siRNA

- SECONDARY siRNAS

Step 4 siRNA amplification by RdRP

More siRNAs = stronger silencing

These new siRNAs load into RISC

they go on to target and degrade more mRNA.

Perfect siRNA pairing

Argonaute cuts the mRNA at the site paired to positions10-11 of the guide siRNA

Cleavage occurs

5′ fragment (uncapped → rapidly degraded)

3′ fragment (no protection → degraded)

Antisense CHS mRNA from the transgene

Antisense CHS mRNA can be converted into double-stranded RNA (dsRNA) by RNA-dependent RNA polymerase (RdRp).

Antisense CHS mRNA can also directly base-pair with endogenous sense CHS mRNA to form dsRNA

Sense CHS mRNA from the transgene

An unusually high level of sense CHS mRNA is recognized by the cell as abnormal

RNA-dependent RNA polymerase (RdRp)uses some of this CHS mRNA as a template to make a complementary antisense strand

As a result, both sense and antisense CHSRNA are present, which pair together to form double-stranded RNA (dsRNA)

recycling of siRNA:

siRNA guide strand remains in the RISC complex and can target multiple mRNA molecules, making the process highly efficient.

siRNA: short-interfering RNA, 21-24 nt

Mostly exogenous origin (i.e., come from external sources). Other than when RdRp makes antisense RNA which then go on to form dsRNAs

- dsRNA precursors

- Target specific

- TRANS-ACTING

siRNA based therapy

Patisiran, givosiran, lumasiran, and inclisiran

Givosiran

drug used to treat acute hepatic porphyria (AHP), a rare disorder caused by overproduction of a protein (enzyme), Aminolevulinic Acid Synthase 1 (ALAS1).

acute hepatic porphyria

Excess of the ALAS1 enzyme is produced --> accumulation of heme intermediates

severe neurological and gastrointestinal symptoms

Givosiran siRNA

siRNA designed to target / bind with perfect complementarity to ALAS1 mRNA

The siRNA binds to the ALAS1 mRNA and the ALAS1 mRNA is destroyed

As a result, less ALAS1 enzyme is produced

Lumasiran

a drug used to treat primary hyperoxaluria type 1(PH1), a rare genetic disorder caused by mutations in the AGXT gene

primary hyperoxaluria type 1 (PH1)

The AGT enzyme activity is absent

This leads to glyoxylate accumulation

Glyoxylate is converted into oxalate

Excess oxalate forms calcium oxalate crystals, which damage the kidneys

Lumasiran siRNA

siRNA designed to target glycolate oxidase mRNA

The siRNA binds to the glycolate oxidase mRNA and it is destroyed

microRNA (miRNA)

1. are always endogenous

2. they originate through TRANSCRIPTION

3. have a stem-loop structure

miRNAs are transcribed from

1. their own independent genes called MIR genes

2. transcribed from specific regions within protein coding genes

3. transcribed from specific regions within non-protein coding genes

Where are microRNAs synthesized fromin the genome?

Intronic miRNA (nc and coding)

exonic miRNA (nc and coding)

miRNAs have specific names

many are conserved across species

- single miRNA (miR-208a) is located inside Intron 27

- Multiple miRNAs (miR-106b, miR-93,miR-25) are located within Intron 13

siRNA function

Functions mainly in defense against foreign double-stranded RNA (such as viruses)

Regulate gene expression when too much mRNA is present

Works by binding perfectly to mRNA and destroying it