Molecular basis of gene expression

What is in the spliceosome

Upp to 5 small nuclear RNAs (snRNAs U1,2,4,5,6)

The formation of tight binding with associated splicing proteins form complexes called snRNPs

How are splice sites identified

Additional sequence info provided by the branch point sequence and the polypyrimidine tract are insufficient in identifying intron and exon boarders

Multicellular eukaryotes needed to encode a number of additional splicing regulators that interact with specific RNA sequences using RNA binding domains

RNA binding regulators

SR proteins, hnRNP proteins and Tissue specific regulators

SR proteins - RNA interaction domain of RRM type (RNA recognition motifs) with a serine and arginine rich domain (SR domain)

hnRNPs and Tissue specific AS regulators - contain RRM aRRM, KH, Glycine rich, Zn finger domain. The large amount of domains increases protein affinity to specific RNA sequences

Splicing silencers and enhancers

The splicing regulators interact with their respective cis elements in pre-mRNAs. The additional regulatory elements are silencers and enhancers. Which repress and promote splicing events respectively

SR proteins often interact with ESEs which are on the majority of exons so SRs control a large amount of splicing events - SR proteins are considered “core splicing machinery”

SR proteins form contacts with U! and U2AF which are the core splicing factors defining the 5’ and 3’ end of introns enabling ESE-bound SRs to stabilize U1 binding to a downstream 5’ss and U2AF to bind to the pyrimidine tract and the 3’ss

PTB1/hnRNP1 can interact with intronic and exonic sequences and often repress the inclusion pf specific exonic sequences into mmRNA

One repression mechanism uses PTB competition for interation sites with core splicing factors and assembly of a multisubunit PTB complex, creates a zone of silencing making the exon invisible to splicing machinery

Alternative splicing

Many pre-mRNAs are spliced in different ways depending on the circumstances, this could include unsplice introns being added or inclusion of different exons.

There are several AS events and these are used to increase protein diversity and to regulate gene expression

Dsx and Drosophila

Thge gene doublesex (Dsx) encodes a transcription factor promoting expression of either male or female specific genes. Dsx contains 6 exons and its pre-mRNA can be spliced in two alternative ways.

Male - exons 1,2, and 3 are joined to 5 and 6 where 6 is polyadenylated at the 3’ end

Female - exons 1,2,3, and 4 assemble the mmRNA where 4 is the last exon that ends ewith the polyA tail

These different processes lead to the different promotion of sexual differentiation all stemming from one gene

Transformer and Transformer-2 (Tra and Tra-2) - Females express both Tra and Tra-2 which biases the splicing choice towards exon 4 terminated mRNA. Males only express Tra-2 which can’t activate exon 4 without Tra so 5 and 6 are used instead

Intricacies of Tra

The Tra gene is transcribed in both males and females but only fenmales produce a functional Tra protein.
The Tra gene has 3 exons - 1 and are constitutive at the beginning and end of the gene respectively. Exons 2 and 3 are a part of the same exon unit appearing short or long (3 or 2-3) depending on the alternative splicing pattern. The longer 2-3 pattern is used in males and only the exon 3 part is used in the female version.

The shorter 3 version is beneficial as the 1,3 and 4 produces an open reading frame making functional Tra proteins in females. The production of functional Tra is impossible from the mRNA with the longer 2-3 as the exon 2 encodes a premature stop codon that interupts the ORF. The male specific Tra therefore produces short versions of the Tra protein that are useless.

Nonsense mediated decay means that mRNAs containing premature stop codons are degraded

The Sxl splicing regulator

The determinant of whether the Tra splice variant is usable or not (which splice variant is made)

The sex lethal gene is expressed in females but not males. It binds to the intronic splicing silencer sequence upstream of the exon 2 part of the 2-3 unit and blocks the use of the exon 2 3’ss encouraging the use of the exon 3 3’ss

Later in development, the early promotter is silenced in both males and females, the Sxl transcription is determined by the late promoter but isn’t sex specific

Thererfore Sxl is handled post transcriptionally - Sxl binds to intronic splicing silencers up and sownstream of the exon 3, skipping the exon ad production of mRNA that has L1 joined with exons 2,4,5,6,7, and 8. This mRNA can produce functional Sxl but if the Sxl was not present in the cell then exon 3 is included which contains a premature stop codon which makes it impossible to make functional Sxl

RNAi

RNA interference silences genes transcriptionally or post transcriptionally

srRNAs can bind to the mRNA targets by complementarty base pairing that can lead to the destruction of the target mRNA or can block its translation.

miRNA

microRNA is a small singles stranded nob-coding RNA molecule containing 21-25 nucleotides from primary miRNA and regulate endogenous gene expression.

At least 60% of human genes are thought to be regulated by miRNAs and some miRNAs can target a few different sites on the same miRNA target with similar nucleotidew sequences

They often bind to the 3’ UTR region of the target mRNA

Primary miRNAs are often greater than 1000 nucleotides in length

Making of miRNA

pri-miRNA is made in the nucleus

A hairpin loop (pre-miRNA) is cleaved from the longer pri-miRNA by Drosha a RNAse III enzyme and a double stranded RNA binding protein called DGCR8

Exportin 5 binds the cleaved pre-miRNA and transports it tp the cytoplasm using GTP bound to a Ran protein

pre-miRNA disengages from the exportin 5 and binds to the Dicer and TAR RNA-binding protein TRBP

Dicer (an RNase III enzyme) cleaves the terminal loop of pre-miRNA to create the miRNA;miRNA duplex that is 21-25 nucleotides long

Dicer carrying the duplex forms a complex with Argonaute (Ago)

The antisense strand is selected and loaded into Ago and the sense strand is discarded

Ago forms a complex woth other proteins called the RISC (RNA-induced silencing complex) and the antisense strand binds to the 3’ UTR of its target mRNA

RNase IIIs

nucleases that are specific to dsRNAand typically contain 1 or 2 RNase III domain and 1 dsRNA-binding domain (dsRBD)

Class 1 - a prokaryote Escherichia coli RNase III - 1 RNase III domain and 1 dsRBD

Class 2 - eukaryote Drosha - 2 RNase III domains and 1 dsRBD

classs 3 - Dicer - same as class 2 with an additional PAZ domain

Dicer

The PAZ domain binds to the 5’ and 3’ ends of the pre-miRNA hairpin and the 2 RNase III domains cut the dsRNA stem and create the duplex

How to identify and characterise miRNAs

• purify miRNA from the total RNA using a size selection method

• ligate 3’ adaptor and 5’ adaptor

• Anneal RT primer and use reverse transcriptase to create ssDNA

• Anneal 2 PCR primers and amplify miRNA

• DNA sequence the PCR products

USING ARRAYS

• synthetic oligonucleotides complementary to different ss-miRNAs are synthesised and spotted on slides

• miRNA is purified from diff tissues and the ends of the miRNA are labelled with fluorescent dye

  • The labelled miRNA is hybridised to oligonucleotides on the slide and the amount of fluorescence is measured

miRNA mutation

miR-96 leading to progressive hearing loss

miR-184 leading to keratoconus and cataract

Abnormal expression of several miRNAs can b einvolved in the formation of cancer

RNA interferne in research

knock down (reduce) the expression of a specific target fene to study the absence of the target gene product on cells or organisms phenotype

Create new antiviral drugs, gene therapies, anti-cancer drugs

The benefit of a degenerate core cis-element is that the possibility to control splicing decisions

U1 and SR proteins define the 5’ splice site and the U”AF binds the pyrimidine tract and the 3’ splice site. Branch point binding proteins recognise the branch site

U2AF is a heterodimer made of a 65Da and 35Da subunit

When bound upstream, it represses U2 snRNP assembly and when bound downstream it activates U1 snRNP assembly