Bio 402/502 Section II, Lecture 4 mRNA processing events and export Dr. Michael C. Yu Lectures powerpoint files & readings (in PDF files) are located in http://biology.buffalo.edu/Faculty/Yu/yu.html Office hours: Weds & Thurs, 9am-12pm, or by appt (email for appointment)

Recap from the last lecture - alternative splicing Alternative splicing: a single gene can encode many messages depending on how the message is spliced What are the molecular mechanisms governing alternative splicing?

Recognition of splice site occurs in pairs 1. Sequence composition of exons and proximity of splice site signals promote exon rather than intron recognition U1 U1 U2AF cryptic 5’ ss GU AG On rate favored because interaction is stabilizing Off rate favored because no stabilizing interaction or it is blocked 2. SR proteins enhances this interaction ESE: exonic splicing enhancer U1 Cryptic splice sites exist adjacent to strong hnRNP binding sites (splicing inhibitors) U1 SR U2AF hnRNP GU AG ESE ISS (Cote, Univ. of Ottawa)

Why are exons preferentially recognized? Differential size distributions of exons (~50 to 300 nt) vs. introns (<100-100,000 nt) SR protein - preferentially binds to exon sequences - mark the 5’ & 3’ splicing sites in conjunction w/ U1 & U2 during transcription hnRNP - heterogenous nuclear ribonucleoproteins (twice the diameter of nucleosome) - consists at least eight different proteins - compacts introns, thereby masking cryptic splicing sites - preferentially binds to introns, but also bind to exons, although less frequently (Cote, Univ. of Ottawa)

Intron Definition Exon Definition Models for splice site recognition: intron & exon definition Boundaries between introns & exons are recognized through its interaction with multiple proteins either across exon or intron Intron definition: Uses intron as the unit of recognition mechanism. Complex forms through stabilized protein interactions across the intron SR SR SR RS 70K U2AF35 RS U2AF RS SF2 Exon 1 A Exon 2 SF1 U1 snRNP Intron Definition SR Exon Definition: Complex can easily form stabilized protein interactions across the exon. Excises out the flanking introns SR RS 70K SR U2AF35 RS U2AF RS SF2 A Exon SF1 U1 snRNP Exon Definition (Cote, Univ. of Ottawa) Stable interaction confirms accuracy of splice site choice

Important point to remember about alternative splicing Regulation of alternative splicing involves the specific stabilization or destabilization of splice site recognition Stabilization: exon inclusion Destabilization: exon skipping (Cote, Univ. of Ottawa)

Approaches used to study alternative splicing How would you identify cis-regulatory sequences responsible for cell-specific splicing ? Mutational analysis finds intronic splicing silencer (ISS) Transfection      Reporter Plasmid   Alternatively spliced Not alternatively spliced Examine RNA Splicing of Transfected Splicing Reporters to identify cis-regulatory regions (Cote, Univ of Ottawa)

Use of deletion analysis to identify intronic splicing silencer First create deletional mutations across a DNA sequence :alternatively spliced exon    RSV  hMT-1 hMT-2 hMT-3 WT D1   WT 101 bp 225 bp deletion  D1 Splicing Reporter Constructs were transfected into the cells and RNA splicing examined by RT-PCR 20% 80% Inclusion D1 contains a sequence element that regulates splicing blue exon (Cote, Univ. of Ottawa)

How can we figure out where it is? ISS allows exon to be skipped   Determining the sequence elements involved in alternative splicing How can we figure out where it is? ISS allows exon to be skipped   RSV ISS  hMT-1 hMT-2 hMT-3 Deletions often guided by conservation of sequence ISS  Exon Skipped (ES) - wild-type 101 bp 225 bp deletion  Exon Incuded (EI) - deletion  ES 1. Deletion arrow the Region to Identify the ISS  ES ISS  EI  ES 2. Point mutagenesis to Identify Key Nucleotides  EI (Cote, Univ. of Ottawa)

UV Irradiate - crosslinks protein to RNA Identifying trans-factors involved in alternative splicing Identify trans-regulatory factors that demonstrate sequence-specific binding. Several Methods: UV Cross-linking, Isolation Assembled RNA/Protein Complexes (Affinity Selection or Gel Shift). Separate & visualize by PAGE UV Cross-linking Assay kDa WT MUT UV Irradiate - crosslinks protein to RNA 220 - 97.4 - Prepare Nuclear Splicing Extract Digest RNA 66 - Normal ISS 46 - Incubate with Normal and Mutant Labeled RNA 30 - Mutant ISS Cell Line (Cote, Univ. of Ottawa) The identity of this protein can be determined candidate approach using antibodies, perform standard protein purification approaches, or affinity approaches followed by microsequencing

Studying alternative splicing using microarrays Comparison: which spliced variants are produced in tissue X vs. tissue Y? Splice variants produced from both tissues Competitive hybridization to special high-definition tiling arrays Verify signals - determine whether exon is skipped/included in a specific tissue type. Can also test splicing changes under specific conditions: nutrients, temperature, response to pharmacological agents, etc (Cote, Univ. of Ottawa)

Overview of mRNA processing steps (McKee & Silver, 2007)

What if the message is not termianted properly? Pre-mRNAs are cleaved and processed at its 3’-end What if the message is not termianted properly? Non-precise termination Precise end after cleavage and polyadenylation AAAAAAAAAAAAA

Pre-mRNA cleavage process involves a set of trans-factors Recognition of target sequences on the RNA by cleavage factors 3’-end cleavage and polyadenylation occur as a single complex Cleavage event provides an indirect signal for transcription termination

Transcription termination model: the allosteric model During transcriptonal elongation, RNA pol II is in a processive/stable conformation As RNA pol II transcribes through the poly A site (AATAAA), it becomes less processive Result: RNA pol II falls off, transcription is terminated

Transcription termination model: the torpedo model After transcribing through the poly A site (AATAAA), downstream RNA sequence is digested by a 5’-3’ exonuclease Once the cleavage occurs, the exonuclease continues degradation of nascent RNA to guide itself to its target--RNA pol II Once it reaches RNA pol II, it dissociates the RNA pol II (torpedoed it!). Transcription is terminated

? Transcription termination model: combined model Cooperation between exonuclease and an unknown helicase or other factors affecting RNA pol II Together, they convert RNA pol II from processive to non-processive form This conversion results in the disruption of DNA:RNA hybrid and releasing of RNA pol II from the DNA template ?

Polyadenylation of pre-mRNAs stabilizes the newly synthesized messages Occurs at the 3’-end of virtually all eukaryotes Only occurs in mRNAs - not in other RNAs produced Polyadenylation is correlated with half-life of mRNA Short poly A = short half-life = little proteins made Long poly A = long half-life = more proteins made

Steps involved in polyadenylation of pre-mRNAs CPSF – cleavage & polyadenylation specificity factor CstF – cleavage stimulation factor CF – cleavage factors PAP – poly(A) polymerase PAB – Poly(A) binding protein G/U – GU or U-rich sequence element CPSF binds to upstream AAUAAA polyadenylation signal in RNA transcript CstF interacts w/ a downstream GU- or U-rich seq., & w/ bound CPSF Physical links between polyadenylation & transcription machinery - CTD of pol II required for efficient capping, splicing, 3’ end processing CPSF co-purifies with TFIID  association of pol II w/ cleavage factors CBC also physically contacts polyadenylation machinery (via assoc. w/CTD) and participates in cleavage All these interactions may stabilize polyadenylation complex Transcription termination dependent on a functional poly(A) signal

AAAAA TTTTTTTT Polyadenylation is useful for isolating mRNA population Cell culture Lyse cells & extract total RNA population mRNAs Chromatograhy column/solid support AAAAA TTTTTTTT Cellulose beads containing poly-dT High temprature: elute mRNAs from the column

RT-PCR of total mRNA population can determine transcription on/off Determining changes with a specific transcript - is it produced under a given condition/treatment? Answers a YES/NO question. RT-PCR: reverse transcribe mRNA (using Oligo-dTs), PCR amplified reverse-transcribed cDNA population, electrophoresis to compare expression No amplification = no mRNA produced Good: detect low levels of expression, only small amounts of mRNA needed Bad: Non-linear comparison, multiple primers needed per gene

A better comparison in terms of relative transcript levels To determine more quantitatively the changes with a specific transcript produced under a given condition: Northern blot/RNA hybridization Steps: isolate total RNA, gel electrophoresis, probe for a specific message (radiolabelled or chemi-illuminescence) Presence/absence of message picked up by hybridizing probe Always normalize the RNA loading by probing for a “control” transcript (Zhang et al, 2000) Not great in comparing small changes in the level of expression

A highly sensitive assay to detect the presence of a specific transcript RNAse Protection Assay - higher sensitivity than Northern blot, but not useful in picking up sizes of target messages Make radioactive labeled RNA from cDNA (your gene of interest) Total mRNA population extracted from cells RNAse treatment removes unpaired probe If a specific mRNA is present, it will “protect” the radiolabelled probe and does not allow it to be digested by RNAse treatment Very sensitive and specific!

Real-time PCR offers quantitative result on # of copies of message Real-time PCR: traditional PCR looks at “end-pt” analysis of products, but real-time PCR allows for detection and quantitation of PCR amplicon throughout a reaction (thus, “real-time”) Theoretically there is a quantitative relationship between the amount of starting sample and the amount of PCR product at any given sample. Real-time PCR detects the accumulation of amplicon during the reaction. The data is then measured at the exponential phase of the PCR reaction rather than end-point plateau. The exponential phase is the optimal point for analyzing data. Area of detection for real-time PCR Exponential: exact doubling of product is accumulating ea cycle w/ 100% reaction efficiency assumption Linear: reaction components are being consumed, reaction is slowing, products are starting to degrade Plateau: end point of PCR (Kapa biosystems)

Real-time PCR: SYBR green method SYBR Green I binds to double- stranded DNA. The resulting DNA- dye-complex absorbs blue light (max = 498 nm) and emits green light (max = 522 nm) As DNA is amplified SYBR fluorescence increases proportionally (Kapa biosystems)

Real-time PCR: Sequence-specific probe (e.g. Taqman) Dual fluorophore-labeled oligonucleotide probe: e.g. TaqMan 5’3’ exonuclease activity of DNA polymerase cleaves reporter dye from quencher and allowing fluorescence. Specific sequences are able to be detected in the real-time reaction. As reporter shifts away from quencher, fluorescence signal is detected (Kapa biosystems)

Information you can learn from total mRNA population Total gene expression using DNA microarrays Able to determine complete gene expression program of a cell at once