Chapter 14

Question 0

RNA splicing terms

Answer 0

Introns are present in genes but not in mature RNA
• How does the information not find its way into mature RNA products of the genes?
– Possibility 1: Introns are never transcribed
• Polymerase somehow jumps from one exon to another
– Possibility 2: Introns are transcribed
• Primary transcript result, an overlarge gene product is cut down
by removing introns
• This is correct process
• Theprocessofcuttingintronsoutofimmature RNAs and stitching together the exons to form the final product is RNA splicing

Question 1

RNA splicing overview

Answer 1

RNA Splicing
• Coding sequence: a series of three-nucleiotide codons that specifies the linear sequence of amino acids in a polypeptide product (contiguous in bacteria and phage genes, but mosaic in eukaryotic genes
• Exon: any region of the primary transcripts that retains in a mature RNA (coding and non-coding exons: non- coding RNAs, microRNAs). Exon does not equal to coding sequence
• Intron: any region of the primary transcripts that is not in a mature RNA (noncoding introns)

Question 2

RNA splicing vs alternative splicing

Answer 2

RNA splicing: precise. (triplet-nucleotide codons of mRNA are translated to in a fixed reading frame of protein coding sequence). If not precise, cause frameshift and result in wrong protein.
• Alternative splicing: different selection of exons can be generated from a given pre-mRNA. These alternative products are called isoforms. Slo gene (encodes a potassium channel expressed in neurons, has the potential to generate 500 alternative versions of that product.

Question 3

Stages of RNA splicing

Answer 3

Stages of RNA Splicing
• Messenger RNA synthesis in eukaryotes occurs in stages
• First stage:
– Synthesis of primary transcript product
– This is an mRNA precursor containing introns copied from the gene if present
– Precursor is part of a pool of heterogeneous nuclear RNAs – hnRNAs
• Second stage:
– mRNA maturation
– Removal of introns in a process called splicing

Question 4

Splicing signals

Answer 4

Splicing Signals
• Splicing must be precise
• Splicing signals in nuclear mRNA precursors are remarkably uniform
– First 2 bases of introns are GU
– Last2areAG
• 5’- and 3’-splice sites have consensus sequences extending beyond
GU and AG motifs
• Branch site (branchpoint site): entirely within the intron, usually close to the 3’ end, and followed by a polypyrimidine tract (Py tract)
• Whole consensus sequences are important to proper splicing
• Abnormal splicing can occur when the consensus sequences are
mutated

Question 5

Mech of splicing (two step model)

Answer 5

Mechanism of Splicing of Nuclear mRNA Precursors
• Intermediate in nuclear mRNA precursor splicing is branched – looks like a lariat
• Two-step model (two transesterification reactions)
– 2’-OH group of adenosine nucleotide in middle of intron attacks phosphodiester bond between 1st exon and G beginning of intron
• Forms loop of the lariat
• Separates first exon from intron
– 3’-OH left at end of 1st exon attacks phosphodiester
bond linking intron to 2nd exon
• Forms the exon-exon phosphodiester bond • Releases intron in lariat form at same time

Question 6

Spliceosomes

Answer 6

Spliceosomes
• Splicing takes place on a particle called a spliceosome
• Yeast spliceosomes and mammalian spliceosomes have sedimentation coefficients of 40S and 60S
• Spliceosomes contain the pre-mRNA
– Along with snRNPs and protein splicing factors
– These recognize key splicing signals and orchestrate the splicing process

Question 7

Splcing: Branch signals

Answer 7

Signal at the Branch
• Along with consensus sequences at 5’- and 3’-ends of nuclear introns, branchpoint consensus sequences also occur
• Yeast sequence invariant: UACUAAC
• Higher eukaryote consensus sequence is more variable
• Branched nucleotide is final A in the sequence

Question 8

snRNPs

Answer 8

Small nuclear RNAs coupled to proteins are abbreviated as snRNPs, small nuclear ribonuclear proteins
• The snRNAs (small nuclear RNAs) can be resolved on a gel:
– U1, U2, U4, U5, U6
– All 5 snRNAs join the spliceosome to play
crucial roles in splicing
RNA-RNA, RNA-protein, protein-protein are important for splicing function of snRNPS.

Question 9

Types of snRNPS

Answer 9

U1 snRNP
• U1 snRNA sequence is complementary to both 5’- and 3’-splice site consensus sequences
– U1 snRNA base-pairs with these splice sites
– Brings the sites together for splicing is too simple an
explanation
• Splicing involves a branch within the intron

Wild-Type and Mutant U1 snRNA
• Geneticexperimentshaveshownthatbasepairing between U1 snRNA and 5’-splice site of mRNA precursor is necessary but not sufficient for binding.
U6 snRNP
• U6 snRNP associates with the 5’-end of the intron by base pairing through the U6 RNA
• The association occurs first prior to formation of lariat intermediate but it persisits after first step in splicing
• The association between U6 and splicing substrate is essential for the splicing process
• U6 also associates with U2 during splicing
U2 snRNP
• U2 snRNA base-pairs with the conserved sequence at the splicing branchpoint
• This base pairing is essential for splicing
• U2 also forms base pairs with U6
– This region is called helix I
– Helps orient snRNPs for splicing
• 5’-end of U2 interacts with 3’-end of U6
– This interaction forms a region called helix II
– This region is important in splicing in mammalian cells, not in
yeast cells
Proteins:
U2AF (U2 auxiliary factor): recognize the Polypyrimidine (Py) tract/3’ splicing site.
BBP (SF1 in mammalian): branchpoint-binding protein
U5 snRNP
• U5 snRNA associates with the last nucleotide in one exon and the first nucleotide of the next exon
• This should result in the two exons lining up for splicing
U4 snRNP
• U4 base-pairs with U6
• Its role seems to be to bind U6
• When U6 is needed in a splicing reaction U4 is removed

Question 10

Canonical splicing pathway

Answer 10

E (early) complex: U1 snRNA recognize 5’ splicing site. U2AF is made up of two subunits, the larger
of which (65) binds to the Py tract and the smaller (35) binds to the 3’ splicing site. The former subunits
interacts with BBP (SP1) and helps that protein bind to the branch site.
A complex: U2 snRNP binds to the branch site,
aided by U2AF and displacing BBP (SP1).
(There is a single nucleotide bulge at the binding
site between U2 and branch site, so that the A residue
is not paired and available to react with the 5’ splicing site.
B complex: brings together all three splicing sites.
The U4 and U6 snRNPs, along with U5 snRNP, joined the complex, form a tri-snRNP particle. U4 and U6 are held together
by complementary base pairing between their RNA components,
and U5 snRNA are loosely associated through protein-protein interactions.
Next U1 leaves the complex, and U6 replaces it at the 5’ splice site. These steps complete the assembly process.

Question 11

Canonical splicing pathway part 2

Answer 11

C (catalysis?) complex: rearrangement occurs. U4 is released from the complex, allows U6 to interact with U2 through RNA:RNA base pairing.
It is striking that not only the active site is formed primarily of RNA, but also that it is only formed at this stage of the spliceosome assembly. (strategy to decrease the chance of aberrant splicing.
Formation of the active site juxtaposes the 5’ splice
site of the pre-mRNA and the branch site, facilitating the first transesterification reaction. The second reaction, between The 5’ and 3’ splicing sites, is aided by the U5 snRNP, which helps bring the two exons together.
The final step is the release of the mRNA product and the snRNPs. The snRNPs can be recycled.
The reaction can go backward and forward. The directionality is ensured because the spliceosome is rapidly disassembled
immediately after the second reaction takes place. A dead-box helicase (Prp22) is involved.

Question 12

Self splicing introns/Group I and II introns

Answer 12

Self-splicing: the intron itself folds into a specific conformation within the precursor RNA and catalyzes the chemistry of its own release. (in the absence of any proteins or other RNA molecules.
Group I introns
• 1. conserved secondary structure. Includes a binding pocket that will accommodate any guanine nucleotide or nucleoside as long as it is a ribose from.
• 2. contains a internal guide sequence (IGS) that base-pairs with the 5-splicing site sequence and thereby determines the precise site at which nucleophilic attack by G nucleotide takes place.
• Group I introns can be converted to ribozymes.
• Early in the evolution of modern organisms, many catalytic functions in the cell were performed by RNAs, and these functions have, on the whole, since been replaced by proteins.
• In group II introns, the chemistry of splicing and RNA intermediates produced are the same as those for nuclear pre-mRNAs

Question 13

What prevents the splicing reaction from reversing itself?

Answer 13

What prevents the splicing reaction from reversing itself?
• Once the group I self-splicing intron has been spliced out, the active site remains intact. What prevents the splicing reaction from reversing itself?
• 1. High concentration of G nucleotides that strongly favors the forward reaction.
• 2. The intron can undergo a further reaction that effectively prevents it from participating in the back reaction. At the extreme 3’ end of the intron is a G, which can bind in the G-binding pocket.
• 3. The 5’ end of the intron can bind along the internal guide sequence. Thus, a third transesterification reaction can occur to circulate the intron.
• 4. The new bond formed with the terminal G is labile and hydrolyzes spontaneously. As a consequence, the intron is relinearized, but is truncated and thus precluded from the back-splicing reaction.

Question 14

How Does the Spliceosome Find the Splice Sites reliably?

Answer 14

How Does the Spliceosome Find the Splice Sites reliably?
Average exon 150nt, average intron ~3000nt.
The average human genes have 7~8 exons, and can be spliced in three alternative forms.
Splice-site recognition is prone to two kinds of errors.

Question 15

Solutions to enhance the accuracy of splice-site selection

Answer 15

Solutions to enhance the accuracy of splice-site selection
• 1. co-transcription splicing: RNA PolII carries the various proteins for RNA processing. When the 5’ splice site is encountered in the newly synthesized RNA, the factors that recognize that site are transferred from the Polymerase carboxy-terminal “tail” onto the RNA. Once in place, the 5’ splice site components are poised to interact with those other factors that bind to the next 3’ splice site to be synthesized. Thus the correct 3’ splice site can be recognized before any competing sites further downstream have been transcribed.
• 2. Exon definition process: SR (serine arginine-rich) protein and ESEs (exonic splicing enhancers). SR proteins bound to the ESE sites recruit the splicing machinery to the nearby splicing site. SR recruits the U2AFs to the 3’ splice site and U1 snRNP to the 5’ site.

Question 16

Variants of splicing

Answer 16

Variants of Splicing
• 1. Trans-splicing: two exons being joined reside on different RNA molecules.
• 2. Alternative splicing: exons can be deliberately skipped, and a given exon is joined to one further downstream.
• 3. Minor spliceosome (AT-AC spliceosome): Use U11 and U12 instead of U1 and U2 (frequency: 1 in 1000 in humans).

Question 17

Trans splicing

Answer 17

Chemistry of the reaction is the same. Difference to cis-splicing:
Form a Y-shaped branch structure.
Rare, but occurs in almost all of the mRNAs
Of trypanosomes (unicellular parasitic flagellate
protozoa).
In C. elegans, all mRNAs undergo trans-splicing
to attach a 5’ leader equence, and many of them undergo cis-splicing as well.

Question 18

Alternative splicing

Answer 18

Alternative Splicing
• Transcripts of many eukaryotic genes (90% of human genes) are subject to alternative splicing
– This splicing can have profound effects on the protein products of a gene
– Can make a difference between:
• Secreted or membrane-bound protein • Activity and inactivity
• Products of 3 genes in sex determination pathway of the fruit fly are subject to alternative splicing

Question 19

Examples of alternative splicing

Answer 19

Is

Question 20

Alternative splicing regulation by activators and inhibitors

Answer 20

ESE or ESS: exonic splicing enhancer or repressor sites
ISE or ISS: intronic splicing enhancer or repressor sites.

Question 21

Splicing activators and silencers

Answer 21

Splicing activator and silencers
• Activator: SR protein (has the arginine and serine rich RS domain and RNA-recognition motif (RRM)). RS domains mediate the interactions between SR protein and the proteins within the splicing machinery.
• Silencer: hnRNPs (heterogenous nuclear ribonucleoprotein). They Include hnRNPA1, hnRNPI (or Py tract binding protein PTB), which bind to RNA, but lack the RS domain, and cannot recruit splicing machinery. Instead by blocking specific splice site, they repress the use of those sites.

Question 22

Two mechanisms of silencer action

Answer 22

HIV TAT Exon3:
SC35 binds to ESE, promote exon inclusion (enhance splicing).
a. HnRNPA1 binds to ESS within the exon, and spreads through cooperative binding until it occludes the ESE and competes off SC35 binding.
b. PTB binds to sequence flanking an exon, and interacts with U1 at the 5’ splice site, which blocks U1 to interact with 3’ splice site components. The U1 at the upstream exon pairs with the U2 at the downstream exon.

Question 23

Alternative Splicing in Drosophila sex determination

Answer 23

SisA and SisB genes are found on the X chromosome and encode transcription activators that control expression of Sxl (Sex-lethal) gene. Sxl has two promoters: Pe (establishment) and Pm
(maintenance). Dpn (deadpan) is found in the autosome (chromosome 2), binds sxl promoter and represses sxl expression. Sis/Dpn ratio is differs in two sexes.

Question 24

Events of Drosophila sex determination

Answer 24

Sxl is a splicing repressor, which directs
the splicing of the RNA transcript from Pm (thanks to the earlier expression from Pe), and ensure that the inhibitory exon is spliced Out (therefore maintaining Sxl expression)
It also regulates the splicing of other female RNAs, like Tra gene, which will generate functional Tra protein in females.
Tra functions as splicing activator, which can regulate the splicing of doublesex (dsx), which can activate female genes.

Question 25

Exon shuffling

Answer 25

Exon shuffling: to produce new proteins during evolution
• Evidence for reshuffling exons:
• 1. The borders between exons and introns within a given gene often
coincide with the boundaries between domains;
• 2. Many genes have apparently arisen from evolution in part via exon duplication and divergence.
• 3. Related exons are sometimes found in otherwise unrelated genes. Intron early model: intron existed in all organisms but were lost from bacteria.
Intron late model: intron never exists in bacteria, but rather arose later in evolution.
Advantage of intron: generate multiple protein products from a single gene.
Allow the exons be divided into several axons, therefore generating the possibility of reshuffling exons.

Question 26

RNA Editing: another way of altering the sequence of an mRNA

Answer 26

Cytidine deaminase: converts C to U.
ADAR: adenosine deaminase acting on RNA, converts A to I (inosine).
Guide RNAs direct the insertion and deletion of Uridines.
Guide RNA:
1, Anchor region: direct gRNA to the region of the mRNA it will edit;
2. Editing region: determine the U insetion pattern
3. Poly-U strech: probably tether guide RNA to the purine-rich sequences in the mRNA upstream of the edited region.
Trypanosome CoxII gene. The insertion of the 4 U nucleotides generate the correct reading frame and coding information of the mRNA

Question 27

mRNA transport

Answer 27

RNA export from the nucleus is a active process, and only certain RNAs are
selected for transport. To be selected
for transport, the RNA must have the correct collection of protein bound to it. These will distinguish it from other RNAs, which must be retained in the nucleus or degrades.
Proteins that recognize exon-intron boundaries, For example, indicate that an mRNA has been appropriately spliced, whereas proteins that bind introns indicate that an RNA that should
be retained in the nucleus. Once in the cytoplasm, some proteins are shed and
others are taken on in readiness for translation.
Export requires energy, which is supplied by hydrolysis of GTP by Ran GTPase. Ran GTPase has GTP and GDP bound state.(The transition from One state to the other drives the movement into and out of the nucleus.)