chapter 18 - RNA splicing and processing

RNA splicing

the process of excising introns from RNA and connecting the exons into a continuous mRNA

Pre-mRNA

the nuclear primary transcript that is processed by modification and splicing to give a messenger RNA (mRNA)

how are introns removed from the nuclear pre-mRNAs

by a large splicing apparatus (spliceosome) that recognizes short consensus sequences concerved within the intron-exon boundaries

what does splicing involve

transesterifications, the catalytic center of the spliceosome includes both RNA and proteins

how can some RNAs excise their introns

autonomously

describe the mRNA transcipt

capped (5’ capping), has introns removed, and is polyadenylated at the 3’ end, before being transported through a nuclear pore to the cytoplasm

hnRNP

the ribnucleoprotein form of hnRNA (heterogenous nuclear RNA), in which the hnRNA is complexed with proteins

when are pre-mRNAs exported

after processing is complete, therefore only found in the nucleus

heterogenous nuclear RNA (hnRNA)

RNA that comprises transcripts of nuclear genes made by RNA polymerase II; it has a wide size distribution and low stability

what does transcription usually start with

dATP or dGTP

the fist nucleotide retains what

its 5’ triphosphate and makes a phosphodiester bond from its 3’ to the next nucelotides 5’

what should happen to mRNA treated with enzymes that should degrade it into individual nucleotides, the 5’ end does not give rise to a dNTP. what happens instead

contains two nucleotides joined by a 5’-5’ triphosphate linkage and also bear a methyl group

what is always the terminal base

a guanine that is added to the original RNA after transcription

how is the 5’ cap formed

by adding a G to the terminal base of the transcript via a 5’-5’ link

important for release from pausing of transcription

the capping process takes place during

in the reverse orientation from all the other nucleotides

new G residue added to the end of the transcript

5’ cap of most mRNA is ______________, but some small noncoding RNAs are _____________

monomethylated, trimethylated

recognized by protein factors to influence mRNA stability, splicing, export, and translation

the cap structure

what are splice sites

the sequences immediately surrounding the exon-intron boundaries. named for their positions relative to the intron

5’ splice site at the 5’ (left) end of the intron includes what

consensus sequence GU

3’ splice site at the 3’ (right) end of the intron includes what

consensus sequence AG

when looking at the process of intron splicing, it is crucial to consider the nature of the splice sites, which is?

the boundaries at both ends of each intron that include the sites of breakage and reunion

how can junctions between exons and introns be determined

comparing the nucleotide sequence of mRNA to the sequence of the original gene

what has well conserved, relatively short consensus sequences

splice sites

what is the GU-AG rule

describes the requirement for these constant dinucleotides at the first 2 and last 2 positions of introns in pre-mRNAs

sequence of a generic intron

GU…………AG

how are the ends of introns defined directionally

the 2 sites have different sequences

GU sequence

5’ splice site, left, or donor site

AG sequence

3’ splice site, right, or acceptor site

what rule do minor introns follow

AU-AC rule with a different set of consensus sequences at the exon-intron boundaries

how do issues with pre-mRNA splicing result

from the simplicity of the splice sites

how does the splicesome ensure the correct pairs of sites are recognized and spliced together

any 5’ splice site can be connected to any 3’ splice site

splice sites are generic

the splicing apparatus is not tissue specific

what does splicing depend on

recognition of pairs of splice sites

functionally equivalent

5’ splice sites and 3’ splice sites

how does splicing temporally coupled with transcription

splicing takes place as the mRNA is being transcribed, before RNA polymerase reaches the end of the gene

a functional splice site has to have the right sequence context. what does that include

specific consensus sequences and surrounding splicing-enhancing elements

what sequence does the splicing apparatus recognize

branch site

what does splicing require

5’ and 3’ splice sites and the branch site

how is a lariat formed

when the intron is cleaved at the 5’ splice site and the 5’ end is joined to a 2’ position at an A at the branch site in the intron

what attacks the bond at the 3’ splice site

the free 3’-OH of exon 1

when is the intron released as a lariat

when it is cleaved at the 3’ splice site

what is transesterifications

a bond is transferred from one location to another

where is the branch site located

18-40 nucleotides upstream of the 3’ splice site

what is the role of the branch site

to identify the nearest 3’ splice site as the target for connection to the 5’ splice site

what are the 5’ and 3’ splice sites and the branch sequence recognized by

components of the spliceosome that assemble to form the large complex

what is the spliceosome composed of

both proteins and RNAs

small nuclear RNAs (snRNAs)

small RNA species confined to the nucleus; several of them are involved in splicing or other RNA processing reactions

ribonucleoprotein particles that include a specific snRNA and its protein partners

snRNAs

small cytoplasmic RNAs (scRNAs)

RNAs that are present in the cytoplasm

what are the 5 snRNAs involved in splicing

U1, U2, U5, U4, U6

usually found as a di-snRNP particle

U4 and U6

________ + additional proteins forms the spliceosome

snRNPs

what is splicing factor

a protein component of the spliceosome that is not part of one of the snRNPs

all snRNPs except U6 contain what

a conserved sequence that binds the Sm proteins that are recognized by antibodies generated in autoimmune disease

what proteins do splicing factors include

proteins required for the assembly of the spliceosome, proteins required for it to bind to the RNA substrate, and proteins involved in constructing an RNA-based center for transesterification reactions

what does recognition of the consensus splicing signals involve

both RNAs and proteins

how does U1 snRNP initiate splicing (first step in splicing)

by binding to the 5’ splice site by means of an RNA-RNA pairing reaction

first complex formed

E complex

what does E complex contain

U1 snRNP, splicing factor U2AF, and SR proteins

what is the E complex converted to (second splicing complex)

A complex when U2 snRNP binds to the branch site

what does the addition of U2 snRNP to the E complex produces

A presplicing complex

what does the A presplicing complex require

ATP hydrolysis and commits the pre-mRNA to the splicing pathway

what is exon definition

complex to from between U2AF at the pyrimidine tract and U1 snRNP at a downstream 5’ splice site

how does the A complex covert to the B1 spliceosome

binding of U5 and U4/U6 snRNPs

what does release of U1 snRNP allow

U6 snRNA to interact with the 5’ splice site and converts the B1 spliceosome to the B2 spliceosome

what happens when U4 dissociates from U6 snRNP

U6 snRNA can pair with U2 snRNA to form the catalytic active site

when can U6 not interact with U2

when U4 and U6 are interacting

what is required to allow spliceosome to proceed to the activated site

U4 release

what happens after mRNA has been synthesized and processed

it is exported from the nucleus to the cytoplasm in a ribonucleoprotein complex

why is transport thought to only occur once splicing is complete

since introns prevent export of mRNA while they are associated with the spliceosome

where does exon junction complex (EJC) assemble and what is it involved in

assembles at exon-exon junctions during splicing, is involved in RNA transport, localization, and degredation

has the ability to exicse themselves from RNA by an autocatalytic splicing event

group I and II introns

what are different modes of alternative splicing

intron retention

Alternative 5’ splice-site selection

Alternative 3’ splice-site selection

Exon inclusion or skipping

Mutually exclusive selection of the alternative exons

how can specific exons or exonic sequences be excluded or included in the mRNA products

by using alternative splicing sites

what does alternative splicing contribute to

stuctural and functional diversity of gene products

where do splicing reactions usually occur

only in cis between splice junctions on the same molecule of RNA

what do all introns include

what does this allow to anticodon to do

a sequence that is complementary to the anticodon of the tRNA

base pair with its intron

what does splicing of tRNA depend on

recognition of a common secondary structure in tRNA

what 2 stages does tRNA splicing occur in

phosphodiester bond cleavage

ligation by RNA ligase

tRNA splicing: one enzyme, three activities

cyclic phosphodiesterase

kinase

ligase

tRNA splicing: what is the 5’-OH end phosphorylated by

a polynucleotide kinase

tRNA splicing: what are the exon ends joined by

RNA ligase

where is RNA pol II able to cease RNA synthesis

terminator regions

what does polyadenylation (creation of polyA tail) protect

the mRNA from degradation

where are the major rRNA encoding genes (18S, 5.8S, and 28S) transcribed by RNA polymerase I

in the nucleolus

what transcribes 5S rRNA genes

RNA polymerase III