What are the in vitro requirements for splicing Group I intron from Tetrahymena?
GTP (or guanosine) and Mg++ and not nuclear extracts (i.e., no proteins)
Why are proteins required for activity of most RNA catalysts?
Presumably to fold the RNA into the appropriate structure.
True or False: Only a few RNAs are sufficient for catalytic activity alone?
True
How are Group I and II introns distinguished?
By different structures and mechanisms of catalysis.
What if perhaps a protein is required for Catalytic RNA splicing but it is resistant to removal by phenol and protease digestion how would you prove it?
In vitro transcribed RNA using purified polymerase had same requirements and spliced at the same correct positions as in vivo and as purified RNA
What is the size of the nt RNA genome of satellite tobacco ring spot virus?
359nt
What virus replicates by rolling circle generating tandem genome repeats?
Satellite Tobacco Ring Spot Virus
What virus folds into a hammerhead structure and is this active or inactive?
Satellite Tobacco Ring Spot Virus and this structure is the ACTIVE form.
True or False: Cleavage of Satellite Tobacco Ring Spot Virus is NOT self catalyzed.
FALSE
How can the cleavage of STRS Virus into monomers be reproduced?
By using two short RNA oligos
RNA oligo 02 on top RNA oligo 01 on bottom. What does this represent?
Two RNA oligos reproducing catalytic activity of STRS virus.
Hammerhead ribozymes made to function in trans (01 causes cleavage of 02 to P1 and P2).
What are the types of alternative splicing?
Simple and Complex
Features of simple alternative splicing?
one promoter, one splicing pattern and one polyadenylation site
Features of complex alternative splicing
multiple promoters, muliple splicing pattterns and poly (A) sites, and multiple opne reading frames (ORFs)
During complex alternative splicing what is the consequence of multi promoters?
Creates diff translation initiation condons (proteins have different N-termini)
True or False: Vetebrate pre-mRNAs do not have incredible sequence complexity?
FALSE
How to avoid recognition of cryptic splice sites?
The 5'SS: AG is well conserved followed by the third g is more you move down the sequence the less conserved the nucleotide sequence becomes. However, splice site sequences do not contain sufficient specificity to distinguish cryptic and bona fide splice sites.
What are the four evidences of exon definition?
1. Mutation of a 5' splice site results in exon skipping, not intron retention.
2. Strengthening a 5' splice site boosts splicing of the upstream intron
3. Exon length effects recognition (too large and too small are problems)
4. Isolated exons assemble a complex indistinguishable from the A complex. Both found to contain U1 and U2 snRNPs and U2AF65/U2AF35.
Mutation of a 5' splice site results in what?
Exon skipping, not intron retention
What causes intron retention?
If splice sites are recognized as pairs across introns, a 5'ss mutation will cause the introns to be retained or use of the "next best" cryptic 5'ss.
Exons are defined by what?
Two splice sites and will be skipped if:
One splice site is mutated or will use an internal cryptic 5'ss as a "defining" 5'ss.
What does strengthening of a 5' splice site do?
Boosts splicing of the upstream intron.
Tested on a weak alternative exon
What lessons are learned from strengthening a 5' splice site?
1. Alternative exons have weak signals
2. improving signals make them constitutive
3. demonstrates functional interaction across exons
True or False: Exon length affects exon recognition (too large and too small are problems)
TRUE
The mechanism of exon definition is partly known and involves a highly conserved family of proteins called _________?
SR proteins
Describe SR proteins
They contain Serine-Arginine rich domain which is now called the RS domain.
The Arginine Serine (RS) repeats of SR proteins act like ________?
Molecular Velcro
Components of teh spliceosome that recognize the 3' splice site and the 5' splice site contain_________.
RS domains which interact with RS domains of SR proteins for cross-exon bridging
What subunit of U2AF recognizes the 3' splice site?
65kDa subunit
What subunit of U1 snRNP recognizes the 5' splice site?
70K
Cross-intron bridging, involved in intron definition of vetebrate introns. What causes this?
SR proteins via the same RS-RS domain interactions
Yeast intron containing genes usually contain ______ intron so there is only intron definition and no ______ definition.
ONE
EXON
SR proteins mediate interaction across exons to do what?
define exons
True or False: SR proteins are evolutionarily conserved protein family (Drosophila to humans)
TRUE
SR proteins contain 1 or 2 _______ and ________ domain of alternating arginines and serines.
RRMS
"RS"
What is phosphorylated in SR proteins?
Serines
What does phosphorylation of SR cause?
Affects intranuclear and nuclear; cytoplasmic distribution and spliceosome assembly.
What is essential for splicing?
SR proteins
SR proteins also complement splicing deficient cytoplasmic extracts
SR proteins promote binding of what proteins to pre-mRNA?
U1
U2AF
U4/5/6
What regulates alternative splicing?
SR proteins
RS domains promote _________ interactions contributing to interactions across exons and introns.
protein:protein
SR proteins binds 5'__________ and exonic _____________.
splice site and exonic splicing enhancers
True or False: SR proteins do not function in nuclear to cytoplasmic transport and mRNA translation.
FALSE
True or False: Exons and introns contain auxiliary splicing elements.
TRUE
True or False: Most if not all exons utilize auxiliary cis-actingelements.
TRUE
Name four catergories of zuxiliary elements.
ESE=exon splicing enhancer
ESS=exon splicing silencer
ISE= intronic splicing enhancer
ISS= intronic splicing silencer
What are the best characterized auxiliary splicing elements?
ESEs
True or False: Computational analysis indicates that most exons are likely to have ESEs.
TRUE
True or False: Mutations within some exons caused cryptic splice site selection.
FALSE
Exon skipping
Describe ESEs
Typically short (6-12nt) variable and found in multiple copies within exons.
SR proteins bind to what and promote exon inclusion?
ESEs
Computational analysis has also identified large numbers of ESSs which do three things...
1. block use of adjacent splice sites
2. function in alternative splicing
suppress potential cryptic splice sites nearby
ESSs bind to hnRNPs H and A1
Exon recognition is a cumulative sum of several features:
1. splice site strength (similarity to consensus)
2. exon lenght (50-200 best)
3. exonic element
4. intronic element
5. strength of competing splice sites
6. secondary structure
True or False: Different exons are recognized in different ways
TRUE
What is trans-splicing?
Intermolecular splicing that joins exons from separate transcripts.
What organsims trans-splice only and what does this mean?
Trypanosomes
All mRNAs contain teh same 39nt leader sequence at the 5' end.
What organisms have both cis and trans splicing?
Nematodes
RNase H digestions in permeabilized Trypanosome cells demonstrated ___, ____ and ____ required for cis-splicing.
U2, U4 and U6
True or False: The mechanism of trans-splicing is essentially the same as cis-splicing.
TRUE
What are the similarites between trans and cis splicing?
They use same two transesterification reactions.
What are the differances between trans and cis splicing?
Because there are two separate RNAs, the reaction produces a branched intermediate rather than a lariat in vitro.
True or False: In nematodes, cis and trans-splicing occurs in the same pre-mRNA
TRUE
Trans and cis spliced introns distinguished by structure, not sequence
True or False: Trans-splicing is used for coordinating gene regulation.
TRUE
About___% of genes in C. elegans are polycistronic?
15%
What sequence in required for SL2 trans-splicing?
AAUAAA (3' forming signal)
downstreams genes have different spliced leader SL2
What is determinative for trans-splicing?
Gene architecture rather than specific sequence
What are two major types of 3' end formation for RNA polymerase II transcripts?
1. Cleavage/polyadenylation (vast majority of genes)
2. replication-dependent histone 3' end cleavage
3' end formation of the vast majority of Pol II (non-histone) genes is a three step process
1. Cleavage of the pre-mRNA
2. Polyadenylation (addition of ~200 adenosine residues to the mRNA 3' end)
3. Transcription termination
Three consensus sequence elements required for cleavage/polyadenylation:
1. AAUAAA strongly conserved in vertebrates and absolutely required
2. G/U-rich region downstream from cleavage site. Quite variable
3. A few genes have upstream activating sequence (UAS) (mostly viral)
Trans-acting factors required for polyadenylation.
3. binding of CPSF to RNA is weak, biochemical purification of stabilizing factor indentified CstF
4. cooperative interactions with CstF the first step of polyadenylation complex assembly, forming stable complex that recruits other factors
5. 73 kDa subunit proposed as endonuclease
6. Required for both cleavage and polyadenylation
Describe four specifics of CstF
1. three subunits 77, 64 and 50 kDa
2. 64 kDa protein binds the RNA; UV crosslinks to the G/U-rich region
3. binding of CsfF to G/U rich region requires AAUAAA, reflecting the cooperative interaction with CPSF
4. CstF 77kDa subunit interacts with the 160 kDA CPSF subunit
What component interacts with the phosphorylated CTD.
Cleavage factors I and II
Called cleavage factos but now thought not to perform the endonuclease cut.
What is PAP?
Poly(A) polymerase that is the distributive addition of poly (A) tail following endonucleolytic cleavage.
First 10-15 A's require factors bound to AAUAAA
Whats the function of PABP?
nuclear protein that binds poly (A) and mediates, in part, teh upper limit of ~200 A's. PABP binds to the growing poly(A) tail and interacts with CPSF and PAP stabilizing PAP binding and allowing rapid A addition. Length control results when these interactions are made difficult by long poly(A) tails.
Be able to identify each and function.
Subunits 100, 30, 73 and 160 CPSF
Subunits 77, 64 and 50kDa CstF
Subunits 25/68 and ? CFII and II
Subunits 82 PAP
What are the roles of the poly(A) tail?
1. binds PABP
2. increases mRNA stability. mRNAs are de-adenylated prior to degradation
3. stimulates translation. PABP interacts with translation initiation factors (eIF4E/eIF4G), stimulates binding of 40S ribosomal subunit to initiate scanning for AUG codon.
4. cytoplasmic polyadenylation is a mech to regulate translation of developmentally important proteins (short tail=no translation; long tail=translated)
What to RNA when PABP and eIF4E were added?
Circular RNA
The polyadenylation complex consist of what components?
CPSF, PAP, PABP
Transcription termination is coupled to ________ in that _________________ are required.
3' end formation
polyadenylation [poly (A)] signals are required
After poly (A) tail is 10nt, what does PABP do?
Stabilizes complex with CPSF and PAP, and converts PAP from distributive enzyme (dissociates after incorporation of each nt) to fully processive elongation to full poly (A) tail of 200-250nt.
After 250nt, PAP becomes distributive again by feedback inhibition of PABP and CPSF
True or False: Splicing enhances polyadenylation and polyadenylation enhances splicing
TRUE
What happens if you mutate the AAUAAA hexanucleotide?
Inhibits removal of the last intron
What happens if you mutate the 3' splice site of the last intron?
inhibits polyadenylation
TRUE or FALSE: The 3' splice site of the last exon enhances polyadenylation efficiency
TRUE
TRUE of FALSE: Mutation of polyadenylation signals reduce splicing efficiency of last intron
TRUE
True or False: RNA processing is cotranscriptional
TRUE
The transition from initiation to elongation during transcription is accompanied by phorphorylation of the CTD by _______, largely on Ser___. More phosphates are added, largely on Ser ___.
TFIIH
Ser5
Ser2
The phosphorylated CTD serves as a docking platform for factors involved in ____, _____ and _____.
splicing, capping and polyadenylation
What is the spatial relationship between transcription and splicing within the nucleus?
The nuclear structures
1. perichormatin fibers (PF)
2. interchormatin granule clusters (IGC)
3. coiled bodies (now called Cajal Bodies) (CB)
Name two facts about perichromatin fibers (PF)
1. contains bulk of newly synthesized pol II RNA by pulse-chase experiments
2. immunofluroescent staining with antibodies to splicing components (SR proteins and snRNP proteins) show diffuse reticular staining that joins IGCs
Name two facts about interchormatin granule clusters
1. No nascent RNA labeled by pulse chase
2. 20-50 immunoflurescent "speckles" per nucleus using antibodies to splicing components and some contain Pol II
Name two facts about coiled bodies (now called Cajal Bodies) (CB)
1. no nascent RNA labeled by pulse chase
2. few intense foci using anti-SR protein antibodies, snRNP
TRUE or FALSE: RNA processing occurs in perichromatin fibrils, probably co-transcriptionally.
TRUE
What is the function of PF and CB?
Could be sites of storage, sites of pre-assembly or recycling.
Antibodies to splicing factors stain IGC ("speckles") in quiescent nuclei. Speckles are thought to be ____________ for splicing and other factors.
"storage areas"
3 main points about RNA editing
non-templated nts generated in mRNA
Mostly in noncoding regions
two types
-insertion/deletion of a base (in/del U)
-base modification (C->U, A->I)
Describe the mitochondrial genome of trypanosomes.
minicircles (encode gRNAs) and maxicircles (encode nonfunctional RNAs)
What is the function of trypanosome gRNAs
transfer sequence info to maxicircle RNA to restore ORF
gRNAs are recycled to multiple mRNAs
Trypansosome have weak RNA-RNA interactions so ________ RNP complex involved (proteins encoded in nuclear DNA)
Editsome
Up to 50% of RNA needs editing to be translatable.
True or False: ADARs have the capacity for unaided site-specific RNA binding and editing.
TRUE
Name four RNase III proteins
Human Drosha
Human Pasha
Human Dicer
Ago proteins (in RISC)
What is included in the miRNA microprocesser complex
Drosha and Pasha
Whats function of Pasha
Stabilized Drosha (prot-prot) interaction. Single Negative Feedback Loop.
What are the components of Ago protein (in RISC) and what does each do?
PAZ-docking for 3' end of small RNA
MID-anchors 5' terminal nt
PIWI-slicer activity (catalytic site)
3 rules for miRNA-mRNA interaction
1. must have perfect bp-ing of highly conserved seed region (nts2-8)
2. bulge/mismatches in central region of duplex prevents cleavage by Ago
3. some complementarity in 3' region of miRNA to stabilize the interaction
3 ways miRNAs regulated?
1. regulation at the processing step
2. regulation by feedback loops
3. change length of 3' UTRs
What enzyme is responsible for 5'-3' mRNA degradation?
XRN1
What enzyme is responsible for 3'-5' mRNA degradation?
Exosome which is a multi-protein complex cable of degrading various types of RNA.
What is the enzyme responsible for mRNA decapping?
DCP2
What are the two mechanisms of mRNA decay?
deadenylation and decapping
Name four types of mRNA decay.
ARE-mediated decay
NMD
miRNA-mediated decay
Fos CRD-mediated decay
What protein recruits CCR4?
c-fos coding region determinant binds UNR which also binds PABP
The exosome is found where and what does it do?
nucleus and cytoplasm
processing and degradation
What do Ski proteins do?
Unwinds secondary structures
2 ways mRNAs are localized?
localized protection of mRNAs from degradation (bind proteins that protect/diffuse RNA degraded)
Cytoskeletal transport of mRNAs (anchor proteins localize mRNAs transported along cytoskeleton)
