RNA Interference (RNAi) and Gene Expression

• Module 7.1 centers around RNA Interference (RNAi)

RNA Interference (RNAi)

• A biological process where RNA molecules halt gene expression
• Achieved by neutralizing targeted mRNA molecules

Interactive Lecture Question 1

• If given an RNA molecule sequence, the template strand will be the sequence that is complementary

Gene Expression

• Gene expression is the result of transcription and translation.
• Transcription involves DNA being transcribed into RNA
• Translation involves RNA being translated into Protein

Small RNA Molecules

• Small RNA molecules are produced in both eukaryotes and prokaryotes
• Messenger RNA (mRNA), Ribosomal RNA (rRNA) and Transfer RNA (tRNA) are produced in both
• Eukaryotes only create Pre-messenger RNA (pre-mRNA), Small nuclear RNA (snRNA), Small nucleolar RNA (snoRNA), MicroRNA (miRNA), Small interfering RNA (siRNA), and Piwi-interacting RNA (piRNA)
• Prokaryotes only produce CRISPR RNA (crRNA)

RNAi and Gene Silencing

• RNAi and post-transcriptional gene silencing
• RNAi condenses chromatin to suppress transcription, therefore mRNA is not made
• in Post-Transcriptional gene silencing, Inhibit translation so protein is not madel
• The mRNA is destroyed before it is translated into protein.

Micro RNA origins and function

• Precursors of miRNAs, called primary miRNA (pri-miRNA), are encoded by the genome and transcribed by RNA Pol II
• In the nucleus, Pri-miRNA is cleaved by Drosha (RNAse III enzyme) into pre-miRNA, then the structure becomes a stem loop.
• In the cytosol, Dicer cleaves the pre-miRNA into 19–25 nucleotide miRNA:miRNA duplex without a stem-loop.

RNA-Induced Silencing Complex

• RNA-Induced Silencing Complex (RISC) contains Argonaute protein
• SIRNA passenger strand is cleaved, complementary binding of the guide strand to target mRNA, resulting in mRNA cleavage
• MiRNA passenger strand is discarded, incomplete complementary binding of the guide strand to target mRNA, resulting in Translational repression, mRNA degradation, and mRNA cleavage

siRNA and miRNA Comparison

• siRNA is composed of double-stranded RNA with up to 100 nucleotides
• miRNA contains Precursor miRNA (pre-miRNA) that contains 70-100 nucleotides with interspersed mismatches and hairpin structure
• siRNA has a 21-23 nucleotide RNA duplex with 2 nucleotides 3' overhang
• miRNA has a 19-25 nucleotide RNA duplex usually with 2 nucleotides 3'overhang
• siRNA is fully complementary to mRNA
• miRNA is partially complementary to mRNA typically targeting the 3' untranslated region of mRNA
• siRNA has 1 mRNA target, miRNA can have multiple mRNA target (could be over 100 at the same time)
• siRNA’s mechanism of gene regulation is Endonucleolytic cleavage of mRNA
• miRNA’s mechanism of gene regulation is Translational repression, Degradation of mRNA, Endonucleolytic cleavage of mRNA (when there is a high level of complementary between miRNA and mRNA)

RNAi and Transcriptional Gene Silencing

• Small RNA (siRNA) duplexes are loaded into a nuclear form of RISC called the RNA-induced transcriptional silencing (RITS) complex
• RITS serves to mediate gene silencing via heterochromatin formation
• RITS are effector complexes that are targeted to homologous sequences by base-pairing interactions involving the guide strand of the small RNA.
• RNAi directs localized repressive chromatin formation

Chromatin structure

• Small RNA molecules form repressive chromatin through histone modification

RNA-induced transcriptional silencing (RITS)

• RNAi mediates histone and DNA methylation
• siRNAs in RITS can base-pair with RNAs from their target DNA
• RITS acts as a recruiter of modifying enzymes to specific DNA regions, catalyzing the methylation of histone tails or cytosine bases in DNA

Why RNAi matters

• Approximately, 30% of human genes are regulated by RNAi, with thousands of genes coding for miRNAs.
• RNAi plays a vital role in regulating gene expression during embryo development.
• RNAi is an important research tool for 'knocking out' (silencing) particular genes.
• RNAi could lead to important advances in treating human diseases

Cancer treatment

• Targeting delivery of siRNA and chemo drugs to cytoplasm of cell is used to treat cancer

• Module 7.2 covers Post-transcriptional Processing and Editing

Colinearity

• Continuous sequence of nucleotides encodes continuous sequence of amino acids
• The number of nucleotides in the gene is proportional to the number on amino acids in the protein
• The colinear model is more accurate for prokaryotes, but not for eukaryotes

Pre-RNA Molecules

• Pre-RNA molecules are produced in both eukaryotes and prokaryotes
• Messenger RNA (mRNA), Ribosomal RNA (rRNA) and Transfer RNA (tRNA) are produced in both
• Eukaryotes only create Pre-messenger RNA (pre-mRNA), Small nuclear RNA (snRNA), Small nucleolar RNA (snoRNA), MicroRNA (miRNA), Small interfering RNA (siRNA), and Piwi-interacting RNA (piRNA)
• Prokaryotes only produce CRISPR RNA (crRNA)

Transcription

• A gene includes DNA sequence that codes for all exons, introns, and those sequences at the beginning and end of the RNA that are not translated into a protein

Prokaryotic mRNA vs Eukaryotic mRNA

• Prokaryotes have a Shine-Dalgarno sequence
• Shine-Dalgarno is in prokaryotes only
• Both have Start Codon, Stop Codon, a 5' untranslated region, a Protein-coding region, and a 3' untranslated region

Prokaryotes Gene Organization

• Prokaryotic protein coding genes are found in a contiguous array in the DNA called an Operon, which operates as a unit
• A single transcription start site is used for multiple genes

Eukaryotic Gene Organization

• Eukaryotic protein coding genes are transcribed each from its own start site, processed into a functional mRNA and encodes a single protein

DNA Noncoding gaps

• Prokaryotic DNA genes contain little to no noncoding gaps (introns)
• DNA is transcribed directly into co linear mRNA, which is translated into protein
• mRNA is still being produced during translation

Pre-mRNA vs mRNA

• Pre-mRNA is combined with a CAP, Start codon, Exon, Intron, and a Poly(A) tail
• Final mRNA is composed of Cap, Start Codon, a Protein-coding sequence, and ends with a UTR

Types of Introns

• Group I introns are for Genes of bacteria, bacteriophages, and eukaryotes are Self-splicing
• Group II introns are for Genes of bacteria, archaea, and eukaryotic organelles are Self-splicing
• Nuclear pre-mRNA’s Protein-encoding genes in the nucleus of eukaryotes are Spliceosomal
• tRNA tRNA genes of bacteria, archaea, and eukaryotes are Enzymatic

Posttranscriptional modifications on mRNA

• 5’ cap addition aids in ribosome binding to mRNA, increases mRNA stability, enhances RNA splicing
• 3 ' cleavage and addition of poly(A) tail Increases mRNA stability, aids mRNA export from nucleus, facilitates ribosome binding to mRNA
• Noncoding introns are removed with RNA splicing during via pre-mRNA, increasing the amount of mRNA, allows for multiple proteins to be produced through alternative splicing
• RNA edition effects the alters nucleotide order of mRNA

Eukaryotic pre-mRNA Processing

• Capping of the 5' end, Polyadenylation of the 3' end, and Splicing (removal) of introns

Caping

• A methylated guanine nucleotide is attached to the 5’-end of the pre-mRNA
• Methyl groups (CH3) are added
• Caping is necessary for efficient initiation of translation, transport of mRNA from nucleus, protects mRNA from degradation, and enhances RNA splicing

Polyadenylation

• Polyadenylation is when 50 to 250 adenine (A) nucleotides are added to the 3'-end of the pre-mRNA.
• Polyadenylation is necessary for efficient translation and protects mRNA from degradation

mRNA splicing

• RNA Splicing removes introns from pre-mRNA
• Three consensus sequences in the pre-mRNA are required; the 5' splice site, 3' splice site and branch point.
• Consensus sequences are used by the spliceosome to recognize and remove introns

Introns removal

• Introns are removed in the form of a lariat to form loop with pre-mRNA
• Exons are spliced together by two successive reactions.

Spliceosome complex

• A Ribonucleoprotein complex [300 proteins and 5 small nuclear RNAs (snRNAs)]

Spliceosome contents

• The spliceosome contains five snRNPs (small nuclear ribonuclear proteins).

• snRNA + protein = snRNP

• snRNP includes U1, U2, U4, U5 and U6

• snRNPs are central to the activity of the spliceosome

• Module 7.3 covers Post-transcriptional Processing and Editing Part 2

Eukaryotic Processing

• Capping of the 5' end, Polyadenylation of the 3' end, and Splicing (removal) of introns

RNA Polymerase

• Functional coupling of mRNA transcription and mRNA processing achieved by RNA polymerase II
• The 'tail' or C-terminal repeat domain (CTD) of the largest subunit of Pol II mediates the coupling
• mRNA processing enzymes are recruited to the CTD of Pol II during transcription.

CAP

• Added when the 5' end of the pre-mRNA emerges from the polymerase
• Capping enzymes recruited to C-terminal domain (CTD) of RNA polymerase (Pol) II during early stages of transcription

Splicing proteins

• Assembly of spliceosome occurs co-transcriptionally while the RNA polymerase (Pol II) is actively transcribing
• Components of the spliceosome are recruited to the RNA while transcription is occurring
• The CTD of Pol II interacts directly with splicing proteins to recruit them to the RNA

Transcription termination

• Remembered from M6.5
• Transcription doesn't end at a specific sequence
• Cleavage of mRNA a specific site is necessary
• A 5'->3' exonuclease degrades exonuclease degrades the remaining mRNA terminating transcription

Transcription termination and Polyadenylation

• Remembered from M7.2
• ~ 50 to 250 adenine (A) nucleotides are added to the 3'-end of the pre-mRNA
• Polyadenylation factors are recruited to the CTD of Pol II. RNA is cleaved at the Poly(A) 3' cleavage site. Degradation of the remaining RNA by Rat1 terminates transcription

Number of proteins

• Can produce 100,000 proteins from 20,000 genes
• The proteome, or the protein complement of the cell, is more complicated than the genome because a single gene can give rise to a number of different proteins

Pre-mRNA

• Can be processed in different ways to produce different mRNA molecules which translates to different proteins.
• Can be processed in two ways; Alternative splicing and the use of Alternative PolyA sites

Alternative Splicing

• Pre-mRNA can produce mRNA through exon skipping, intron retention, alternative splicing, mutually exclusive eons

Disease

• Mutations that affect pre-mRNA splicing, Affect use of splice sites, affect the splicing machinery, and affect regulators of alternative splicing can result in many genetic diseases
• 15% of Single nucleotide mutations can lead to diseases that alter pre-mRNA splicing
• Mutations alter pre-mRNA splicing disrupt consensus sequence, create a new 5’or 3’site, initiate usage of an existing cryptic 5’ or 3’site
• Mutations in exon/intron that generate non functional protein or alter RNA stability contribute to disease

RNA Editing

• A gene can be found with a sequence of nucleotides that doesn’t exactly match RNA molecule. Editing alters the coding information of an mRNA transcript
• Edited through Substitution or Insertion
• Chemical alteration of individual nucleotides by specific enzymes is done in substitution editing
• An example is where cytosine is converted to Uracil
• Insertion
• U nucleotides can be inserted into the cell by reacting w complex enzymes
• U nucleotides are catalyzed under the direction of guide RNA or gRNA and can base pair with the mRNA template to add/template for the addition of nucleotides

Eukaryotic RNA

• Is processed in the cytoplasm and before exporting and translation occurs
• A modified nucleotide is added to the 5’ end and a poly-A tail is added to the 3’ end to initiate(capping) and enhance (polyadenylation) process
• Introns of non coding sequences like exons are removed and coded sequences are combined via splicing
• Splicing takes place in the center of Ribo nucleoprotein complex- spliceosome