Topic 8: Posttranscriptional Gene Regulation

Where is newly transcribed precursor mRNA (pre-mRNA) processed to translatable mature mRNA in eukaryotes?

Nucleus

5’-UTRs and 3’-UTRs are part of what?

The first and last exon, and are untranslated regions

What do UTRs do?

Regulate mRNA stability and translational initiation

When is the 5’-CAP and 3’ poly-A tail added?

5’-CAP is added early in transcription and 3’ poly-A tail is added soon after cleavage of mRNA by CPSF

What does pre-mRNA contain?

Alternating coding segments and UTRs (exons) and non-coding segments (introns)

What does the removal of introns by splicing during transcription do?

Generates the open reading frame (ORF) consisting of a continuous stretch of codons (flanked by start and stop codons) and the UTRs

What is RNA processing coordinated by?

Recruitment of enzymes to CTD of RNA pol II

Once processed, where is mRNA transported?

Exported from nucleus into cytoplasm to associate with ribosomes (for translation)

What does the 5’-CAP contain?

7-methylguanosine (7-MeG) is added to 5’ end of pre-mRNA via 5’,5’ triphosphate linkage by guanylyltransferase after the first 20-30 nt of the transcript have been added

What does 5’-CAP do?

• Protects mRNA from degradation by 5’-3’ exoribonucleases because a 5’ phosphate has been replaced with a methylated guanosine (steric hindrance)

• Also acts as a ribosome binding site whereby the 5’ cap recruits the cap binding complex (CBC)

What does the CBC do?

Helps recruit a small ribosomal subunit (40S) to the mRNA which then scans for the start codon (and to the right end)

What did the classical studies of 5’ cap do?

Synthesized 5’-capped and uncapped mRNA by transcription assay using wheat cell extracts with labels labeled GTP and SAM (produces 7-meG)

• sucrose density gradient centrifugation showed that only 5’-capped mRNAs were bound to ribosomes

What is a poly A tail?

String of adenines added to 3’ end of eukaryotic mRNA by poly-A-pol (PAP)

What does the poly A tail do?

Protects mRNA from degradation and promotes translation 

What binds to the poly A tail and protects it from degradation by 3’-5’ exonuxleases?

Poly A binding protein (PABP)

What else does PABP bind?

Eukaryotic translational initiation factors (eIFs) at 5’ cap

What does PABP (3’) and eIF (5’) binding create?

Creates a “closed-loop'“ interaction which brings 5’ and 3’ ends of mRNA together

• this helps the 40S ribosomal subunit bind to the mRNAs 5’ end and begin translation more efficiently

• increases the stability of mRNA

How does alternative splicing give rise to isoforms?

Splicing can occur in different combinations to generate two or more different mature mRNAs from a gene, which creates several related protein products (isoforms)

What are spliced out and what are kept?

Introns are spliced out, and certain exons are selected for inclusion

What are alternative polyadenylation sites?

Alternative polyadenylation (APA) sites that contain variant sequences that bind to CPSF (cleaves AUAA site) as well as other sequences such as GU-rich regions that determine choice of site

What do APA sites do?

Change lengths of 3’-UTR which affects stability, translation and localization of mature mRNA

What is the importance of isoforms created by alternative splicing and APA sites?

A single gene can produce different isoforms in different tissues generating tissue-specific phenotypes

• Increases the number and variety of proteins (proteome) that can be encoded by the condensed genome

• Minimizes # of genes and allows genome to stay compact

What are the 4 features of introns?

1. 5’ splice site sequence

2. 3’ splice site sequence + polypyrimidine tract

3. Begin with GU and end with AG (consensus sequences needed for efficient splicing)

4. Branch point site: located close to 3’ end of intron and is followed by the polypyrimidine tract

How are introns removed?

Two transesterification reactions where two phosphodiester linkages within pre-mRNA are broken and two new ones are formed (energetically neutral)

What is the first step in splicing the intron?

2’-OH of conserved A at branch site attacks the conserved G in the 5’ splice site resulting in an extra 5’-2’ phosphodiester bond (A has three phosphodiester bonds)

What is the second step in splicing the intron?

Phosphodiester bond between the sugar and the phosphate at the 5’ junction between the intron and exon is cleaved and the freed 5’ end of the intron is joined to the A within the branch point

What is the last step in splicing the intron?

The 5’ exon is freed and its 3’-OH attacks the phosphoryl group at the 3’ splice site of the intron joining the 5’ and 3’ exons

• intron is liberated as a lariat and then rapidly degraded

What performs the splice reaction?

The spliceosome, consisting of 5 small nuclear ribonucleoproteins or snRNPs

What does each snRNP contain?

Small nuclear RNA (snRNA): U1, U2, U3, U4, U5, and U6 that form 2 prime structures

What do the snRNAs bind?

Sm proteins at the Sm-binding site near the hairpin loop at the 3’ end and other proteins unique to each snRNA

The spliceosome assembles from snRNPs and other proteins not associated with sRNAs such as….

SR (serine/arginine-rich) proteins that select and regulate splice sites in pre-mRNAs

What does selection of splice sites depend on?

Flanking sequences of the splice sites and branch point, as well as tissue specific splicing factors

How is correct splice site selected?

Base pairing between snRNA and pre-mRNA 

What does spliceosome assembly on pre-mRNAs involve?

Base pairing to snRNAs

What is step #1 in spliceosome assembly?

U1 snRNP binds to GU at 5’ splice site and accessory protein U2AF binds AG and flanking sequence at 3’ splice site

What does U2AF then recruit?

U2 snRNP to the branch point

What binds to U2 at the branch site?

U6 of the U4-U6-U5 trimeric snRNP

What occurs after U6 of trimeric snRNP binds U2?

U1 is released, U5 base pairs with 5’ exon and U6 moves to base pair with 5’ splice site, then U4 is released

What happens after U4 is released?

U2 and U6 snRNPs catalyze nucleophilic attack of the 2’-OH of the branch point adenosine at the 5’ splice site cleaving the 5’ exon-intron junction and shifting U5 snRNP to the 3’ splice site

What happens after first nucleophilic attack?

U2-U6-U5 complex remains bound to intron lariat and catalyzes nucleophilic attack of the 3’-OH of the 5’ exon on the phosphodiester bind linking the intron to the 3’ exon

What happens last?

Lariat is cleaved and exons are joined

Why is ATP required for the splice reaction?

Required for unwinding of the snRNAs and proteins and snRNA base pairing with each other and the pre-mRNA

Where are self-splicing introns found?

Eukaryotes, prokaryotes and bacterial viruses

What are group 1 self splicing introns?

Requires a free guanine nucleoside whereby its 3’-OH forms a 3’-5’ phosphodiester bond with the 5’ end of the intron and releases the 3’ end of the first exon

• Then the 3’-OH of the first exon attacks the 3'-end of the intron, releasing it and then joining the two exons together

What are group 2 self splicing introns?

Self-splicing reaction is like spliceosomal splicing (lariat formation) and depends on RNA structure to create an active site for catalysis

• 6 domains whereby 1 contains binding sites for 5’ and 3’ splice sites and 6 contains the adenosine to initiate the splicing reaction

Why did RNA likely come before DNA?

RNA stores genetic information AND can also catalyze reactions like an enzyme

• DNA developed later with advantages like increased stability, double stranded allows for complementary strand to be used as a template to repair damaged strand

What level is silencing at?

Posttranscriptional level (mRNA degradation and translation repression)

Where was silencing first observed?

Petunias where overexpression of a pigmentation gene resulted in its own downregulation (co-suppression of gene expression)

How are siRNAs generated?

Long dsRNAs are cleaved to 21-23 bp by the Dicer Rnase III

What is done once siRNAs are generated?

siRNAs are unwound and one of the strands functions as a template in RISC (RNA induced-silencing complex) to guide cleavage of the complementary mRNA

• hunts down complementary sequence to stop translation → gene silenced

What is the purpose of chemically synthesizing siRNAs?

Partial LOF of a target gene

• NOT 100% LOF, since not all mRNA will be degraded

What is gene silencing thought to have originated from?

Ubiquitous in almost all eukaryotes (developed early on in evolution)

• thought to have been an ancestral antiviral mechanism to destroy viral dsRNA (viral RNA genome → dsRNA intermediates → Dicer Rnase III produces siRNA for RISC to use to stop viral RNA translation)

What are microRNAs?

Endogenous gene silencing regulatory RNAs

• noncoding RNAs located in intergenic regions or within genes are transcribed by RNA pol II

What is the difference between siRNAs and microRNAs?

siRNAs are “foreign” whereas microRNAs are actually within the genome

Where are ssRNA (pri-miRNAs) cleaved?

Cleaved in the nucleus by Drosha generating a short hairpin mRNA (pre-miRNA) which is then processed by Dicer and RISC complex like siRNAs

What happens if there is high complementary between miRNA and mRNA?

mRNA is degraded by ribonucleases

What happens if there is lower complementary between mRNA and miRNA?

mRNA is translationally repressed and then eventually degraded (translation initiation factors replaced with mRNA decay factors)

What does CRISPR-Cas9 contain and what does it do?

Dual RNA molecules that guides DNA endonuclease (Cas9) to direct DNA cleavage (RuvC1 and HNH each cut one strand) at specific site in the genome

• genetic engineering in unicellular and mutlicellular eukaryotes, as it allows for precise insertion, deletion, and tagging of genes

• Genetic manipulation is permanent and heritable, and there is no transgene (eg antibiotic resistance; foreign DNA) remaining in mutant like seen in homologous recombination

For CRISPR-Cas9, where do the spacers originate?

From plasmid and viral sequences that once infected the bacteria, its like a record of past infection

• In the CRISPR locus, there is a Cas9 operon, which is followed by the repeat-spacer array

What are the spacers transcribed to?

Transcribed to form crRNA which bind Cas endonucleases and direct them to cleave foreign DNA sequences by complementary base pairing

• bacterio-adapted immune system

What does crRNA do?

Contains a repeated portion and an invader targeting portion (protospacer)

Why is tracrRNA required?

Required for crRNA maturation by forming a tracrRNA:crRNA duplex that is recognized by Cas9

How is RNA-guided Cas9 directed to target DNA?

Complementary base pairing of a 20 nt sequence in mature crRNA

• both strands are cut if Cas9 is fully functioning

What does DNA targeting require?

A PAM sequence (5’-NGG-3’) which is required for initial binding of DNA target to Cas9

What is a single guide RNA (sgRNA) and what two features does it have?

Combine dual tracrRNA:crRNA to sgRNA that have:

• 20 nt sequence at 5’-end and dsRNA at 3’ end that is recognized by Cas9

  • 20 nt of the sgRNA can be used to program CRISPR-Cas9 to target any DNA sequence IF it is adjacent to a PAM

When CRISPR-Cas9 creates a ds break, which two pathways can repair it and what does it create?

1) Non-homologous end joining (NHEJ): error prone and introduces indels

• used to create LOF alleles

2) Homologous recombination uses the wild-type copy of the chromosome or donor DNA for gene correction

• greater efficiency if nick is made by a defective Cas9 where one of the endonuclease subunits is nonfunctional

What is the only approved CRISPR based therapy so far?

Sickle cell anemia and beta thalassemia caused by a non-funcitonal beta hemoglobin gene

What gene is a transcriptional repressor of fetal gamma hemglobin?

BCL11A gene

What does CTX001 (Casgevy) do?

Inactivates BCL11A by CRISPR-Cas9 in extracted blood stem cells allowing production of gamma hemoglobin in place of beta hemoglobin

• CRISPR-edited cells are then transferred into patient

• $2.2 million USD for curative therapy