Exam 3: Ch 8 Pre-mRNA Processing & Transport Across Nuclear Envelope Flashcards
functional RNAs are exported to the cytoplasm as components of _____________
ribonucleoproteins
for protein coding genes, the amount of protein produced depends on 2 things
stability of the mRNAs in the cytoplasm
rate of their translation
are cytokine mRNAs unstable?
yes
why are mRNAs encoding proteins required in large amounts extremely stable
so multiple proteins can be transcribed from each mRNA
miRNA
micro RNA: ~22 bp long that base pair extensively, but not completely with mRNA
regulate stability and inhibit translation of target mRNAs
RNAi
RNA interference
leads to degradation of RNA using siRNA
viral RNA in infected cells
degradation of transposon-encoded RNA
siRNA
short interfering RNA: ~22 bp long that are perfectly complementary to an mRNA sequence
inhibit translation of specific mRNA through RNA knockdown
cleave target RNA, leading to rapid degradation
RNA knockdown
using siRNA to inhibit translation of a specific mRNA by degradation
post-transcriptional gene control
all the mechanisms that regulate gene expression after transcription
includes stability and translation rate
hnRNA
heterogeneous nuclear RNA
include pre-mRNA and RNA processing intermediates with 1+ introns
snRNA
5 small nuclear RNAs that function in removing introns from pre-mRNA by RNA splicing
2 small nuclear RNAs that substitute for the 1st two at rare introns
pre-tRNA
tRNA precursor has more transcribed bases at the ends than mature tRNA
may have an intron in the anti-codon loop
pre-rRNA
long rRNA precursor that is processed into mature rRNA
cleavage, removing bases from ends of cleaved product, modification of bases
snoRNA
small nucleolar RNA
base pair with complementary regions of pre RNA
directs cleavage of RNA chain and base modification
order of RNA processing
capping
polyadenylation
splicing
why is RNA processing important
protect pre-mRNA from enzymes that digest uncapped RNAs
co-transcriptional
pre-mRNA processing occurs in nucleus while nascent mRNA is being transcribed
ribonucleoprotein (RNP) complexes
proteins that are always associated with mRNAs
order of RNP complexes
pre-mRNP
nuclear mRNP
cytoplasmic mRNP
when is the 5’ cap added to nascent RNAs
after transcription initiation
purpose of 5’ cap
mark RNA as pre-mRNA to protect from digestion by enzymes
hnRNP
heterogeneous ribonucleoprotein particles
nuclear proteins + hnRNA
help with RNA processing: splicing, polyadenylation, export through nuclear pores to cytoplasm
how were hnRNP proteins discovered?
UV radiation causes cross-links btw RNA bases and proteins
chromatography recovered proteins
monoclonal Ab specific for major proteins
can hnRNP binding to pre-mRNA make it more accessible?
yes
are there specific hnRNPs in the nucleus and in the cytoplasm?
yes
RNA recognition motif (RRM), RNP motif, RNA-binding domain (RBD)
found in hnRNPs and other RNA-binding proteins
KH motif
found in hnRNP K proteins and other RNA-binding proteins
splicing in short vs. long transcription units
short: follows cleavage and polyadenylation of pre-mRNA
long: starts before transcription of gene is done
which RNA sequences are retained in mature mRNA
near 5’ cap and near poly A tail
how were introns discovered
electron microscopy of RNA-DNA hybrids of adenovirus DNA and the mRNA encoding hexon
splice-site
exon-intron junctions
during splicing _____ base pair with pre-mRNA
snRNA U1, 2, 4, 5, 6 and ~6 proteins each
~170 proteins
U1 binds to which part of pre-mRNA for splicing
5’ end
spliceosome
a large RNP complex made of the 5 splicing snRNPs and other proteins on a pre-mRNA
~170 proteins and ~100 splicing factors
consensus sequence
a moderately conserved short sequence at splice sites flanking introns in pre-mRNA
splicing occurs at short, conserved sequences in pre-mRNA via 2 _____________ rxns
transesterification
in transesterification splicing, introns are removed as ______ structures
lariat (lasso) intermediate
5’ G of intron joined to adenosine (A residue) near 3’ end
branch point
the adenosine residue in the lariat structure that forms an RNA branch
exon-junction complex
after RNA splicing a group of hnRNP proteins stays bound to spliced RNA 20 nt away from each exon-exon junction
one of the hnRNPs is REF
REF
RNA export factor
an hnRNP that helps export processed mRNP from uclus to cytoplasm
nonsense-mediated decay
hnRNPS that help degrade wrongly spliced mRNAs
trans-splicing
construction on mRNA by splicing together separate RNA molecules
occurs in 2 types of protozoans
uses snRNPs
CTD
carboxyl termianl domain
found in RNA pol II
very long
exonic splicing enhancers (ESE)
sequences within exons that show where the splice sites are
SR proteins interact
SR protein
family of RNA-binding hnRNPs that interact with (ESEs) with an RRM RNA-binding domain
long stretches of protein-protein interaction domains of serine and arginine (RS) domains
cross-exon recognition complex
complex of SR proteins, snRNPs and other splicing factors that assemble on an exon
allows for precise specification of exons in long pre-mRNAs
self-splicing
some introns are removed on their own in nonphysiologcal test tube conditions
group I introns in rRNA genes of protozoans
group II introns in mitochondria/chloroplasts
which domains in group II introns can be trans-acting and like snRNAs and why is this important
domain I and V
shows that group II introns may have evolved into trans-acting snRNAs that perform the same function (splicing)
do snRNPs stabilize the geometry of snRNAs and intron nucleotides required to catalyze pre-mRNA splicing?
yes, in theory
which mRNAs dont have a poly A tail
histone mRNAs
early studies of pulse-labeled adenovirus and SV40 RNA demonstrated…
that A residues are added to a 3’ OH produced from cleavage of a longer transcript
3’ cleavage and polyadenylation uses…
poly(A) polymerase (PAP) binds to complex before cleavage can occur
multiple poly(A) binding protein copies (PABPII) adds to short poly A tail added by PAP
what degrades RNA introns that are processed out of pre-mRNAs and improperly spliced/polyadenylated pre-mRNAs?
nuclear exoucleases through hydrolysis
11 of these exonucleases form an exosome (helicases too)
nuclear cap binding complex
protects 5’ cap from exosome degradation
alternative splicing generates…
transcripts with different combinations of exons
make isoform proteins (ex. fibronectin)
a cascade of regulated RNA splicing controls fruit fly ____________ __________
sexual differentiation
what is Sx1 protein encoded by sex-lethal gene
Sx1 found only in females binds to a pre-mRNA and allows splicing of exon 3 (males keep exon 3 which codes stop)
Sx1 proteins allows splicing of transformer gene pre-mRNA
transformer (Tra) protein
encoded by transformer gene in female drosophilia
allows processing of pre-mRNA from double-sex gene –> Dsx protein
male vs. female Dsx protein
longer than female
transcriptional repressor that inhibits expression of genes for female development
female represses genes for male development
splicing _______ and _________ control splicing at alternative sites
repressors, enhancers
ex. fruit flies Dsx protein / fibronectin by hepatocytes vs. fibroblasts
RNA binding sites for splicing repressors are usually
hnRNPs
can also occur in exons: exonic splicing silencers
RNA binding sites for splicing enhancers are usually
SR proteins
can also occur in introns: intronic splicing enhancers
complex regulation of alternative RNA splicing through ____-_______ modifications of ______ factors play a significant role in modulating _____ ______
post-translational, splicing factors
neuron function
ex. Ca activated K channel in hair cells
RNA editing
a type of pre-mRNA processing where the sequence of a pre-mRNA is altered, changing the mature mRNA
mitochondria of protozoans/plants and chloroplasts
apoB gene
encodes two alternative forms of a serum protein for uptaking and transporting cholesterol
apoB-100 (liver) and apoB-48 (intestine)
defect can lead to atherosclerosis
LDL has apoB___ on its surface
apoB-100
deliver cholesterol to tissues by binding LDLr
RNA editing in mammals
changes apoB pre-mRNA to synthesize a shorter apoB-48
the cell-type specific expression of the 2 types of apoB result from editing of apoB pre-mRNA (intestinal only)
fully processed mRNAs in nucleus are bound to hnRNPs in complexes called…
nuclear mRNPs
before an mRNA can be translated, it must be exported out of the _____ to the _____
nucleus, cytoplasm
nuclear pore complex (NPC)
large, symmetrical structures made of 30 different proteins called nucleoporins
FG-nucleoporin
line the central channel through the NPC
semi-permeable barrier lets small molecules only diffuse
mRNPs are transported through the NPC by…
mRNP exporter
uses REF (RNA export factor), a component of the exon-junction complex
also uses SR proteins
direction of mRNP export from the nucleus is controlled by…
phosphorylation and dephosphorylation of mRNP adapter proteins like REF
pre-mRNAs in _____ are not exported from the nucleus
spliceosomes
incompletely spliced mRNAs associate with hnRNPs in the spliceosomes
HIV ___ protein regulates the transport of unspliced viral mRNAs
REV protein
constitutive transport element (CTE) in retroviruses other than HIV
binds to mRNP exporter to allow transport of unspliced retroviral RNA
Rev protein
binds to Rev-response element (RRE) and allows unspliced and singly spliced HIV mRNAs through the NPC
