post transcriptional regulation of gene expression
poly(A) signal
AAUAAA 10-35 nt upstream of cleavage site where CPSF binds
pyrimidine-rich region
AKA poly-pyrimidine tract roughly 15 b long rich in u and c
CSTF
cleavage stimulation factor binds to another cis-acting element downstream sequence which is GU or U Rich
RNA editing is rare in
rare in vertebrates (involves deaminase enzymes)
uses of poly a tail in lab
1. ribosomal and tRNAs predominate in cells 2. mRNAs can be purified from cellular population of RNAs by passing through an oligo dT column 3. only RNA polym II transcripts have 5' cap and 3' tails
gene expression can be regulated at these steps
1. transcriptional control 2. RNA processing control 3. RNA transport control 4. translational control 5. mRNA degradation control 6. protein activity control
functions of snurps
1.mediate RNA splicing 2. cleavage of histone RNA 3' end 3. active area of research
step 2 two step enzymatic reaction of splicing
3' -OH end of first exon adds to the beginning of second exon sequence, cleaving the RNA molecule at the 3' splice site then the two exons are joined
RNA splicing requires
ATP U1, U2, U5, and U4/U6 snRNPs in addition to other proteins in cell extract
example of RNA editing
Mammalian Apolipoprotein B (apoB) gene can produce two alternative forms of proteins both forms are part of lipid complexes that transport lipids in the serum and results from editing of pre-mRNA in the nucleus
PABP II signals
PAP to stop and rapid degradation of uncapped downstream RNA occurs after cleavage
primary transcripts
RNA molecules freshly synthesized by RNA polymerase II in the nucleus that are modified covalently at 5' and 3' ends
precision of RNA splicing
RNA processing machinery guarantees that each 5' splice site pairs only with the 3' splice site closest to it point mutations can inactivate a splice site
snRNP proteins single Ab recognizes
U1, U2, U5, and U4/U6 snRNPs
hnRNA
a collective term referring to pre-mRNA and other nuclear RNAs of varying sizes
primary transcript
a faithful copy of the gene containing both exons and introns
Splicesome Complex
a large multicomponent ribonucleoprotein complex in which U snurps are part of
5' cap addition
add 7-methyl G nucleotide almost immediately after 25-30 nucleotide synthesis which occurs co-transcriptional only polymerase II products get capped it is important in initiation of protein synthesis and RNA stability
Eukaryotic RNA processing
addition of 7-methyl guanosine cap RNA splicing cleavage polyadenylation
hnRNP proteins
are the major protein components of hnRNPs e.g. hnRNP A1, hnRNP C, hnRNP H mostly RNA binding proteins are involved
systemic lupus erthematosus
autoimmune disease where proteins make antibodies against one or more of their own proteins
only LDL complexes (which contian apoB-100) can
can deliver cholesterol to body tissues by binding LDL receptors of cells this is important in the pathogenic processes of atherosclerosis
Thalassemia syndromes
caused by single nucleotide changes (point mutations) that can inactivate a splice site loss of splice site causes recognition of new "cryptic" site near by resulting in altered proteins changes in splicing pattern caused by random mutations could be an important pathway in the evolution of genes and organisms
RNA splicing
cutting out of intron sequences from primary transcripts and joining of exons sequences to produce mRNA molecule that codes directly for a protein it occurs in the nucleus and RNA is exported to the cytoplasm after it is complete
hnRNP particles for very quickly at specific RNA sequences
e.g. splicesome complex that catalyzes RNA splicing
hnRNP particles
heterogeneous nuclear ribonucleoprotein particles which contain hnRNAs and hnRNP proteins newly synthesized RNA in eukaryotic cells becomes condensed into a string of closely spaced protein-RNA containing particles ~500nt RNA wrapped around a protein complex (20nm)
RNA splicing diagram
highly conserved 5' splice site (donor side) and 3' splice site (acceptor side) sequences need for precision reaction for breaking and rejoining of RNA to prevent frame shifts info obtained mainly from in vitro studies
Introns are also known as
intervening sequences
RNA editing
is a unique type of pre-mRNA processing where pre-mRNA and mature RNA sequence is altered
in mammalian cells only about 1.5% of DNA sequence
is copied into functional mRNA sequences (a majority of DNA is now considered to be transcribed to some type of RNA)
functions of poly A tail
mRNA export from nucleus to cytoplasm, stability, efficiency of translation, recognition signal for ribosome, make sure that RNA is intact
RNA editing is common in
mitochondria of protozoans and chloroplasts
RNA polymerase III
moderately sensitive to amantin transcribes 5s rRNA (ribosomal component) tRNAs (protein synthesis) a variety of very small and stable RNAs e.g. U6 (snRNA) and 75 RNA (component of SRP)
the two levels of selection in eukaryotic cells
only a part of the DNA is transcribed and minor proportion of sequences in nuclear RNAs survive RNA processing events
RNA Polym I
polymerase that is unaffected by alpha amantin poison (amantia phalloides) and transcribes pre-rRNAs that are processed into- 28s rRNA, 18s rRNA and 5.85s rRNA: as ribosomal components for protein synthesis
RNA polymerase II
polymerase that is very sensitive to alpha amantin and transcribes genes into mRNAs for protein production most snRNAs (4/5): U1, U2, U4, U5 for RNA splicing siRNAs: for chromatin-mediated repression and translation control miRNAs: for mRNA stability/translational control
Trypanosomes
potential drug targets for pathogenic protozoans in order to do this U's are added or deleted following base-paired short guide RNAs encoded by thousands of mini-mitochondrial DNA circles
cleavage-adenylation
proteins that are part of this complex are CFI (cleavage factor I) and CFII poly (A) polymerase (PAP)
Mechanism of RNA splicing
recognition by complementary base pairing of U1 RNA and U1 snurp to 5' splice site (9nt) recognition of branch point region by U2
is different from that of genomic exons
sequence of mature, processed RNA
snRNPs
small nuclear ribonucleoproteins complexes of proteins with small nuclear RNAs (snRNAs) 'U' rich: U1, U2, U5, and U4/U6
step 1 two step enzymatic reaction of splicing
the branch point A nucleotide in the intron (located near the 3' splice site) attacks the 5' splice site and cleaves it then, the cut 5' end becomes covalently attached to this A to form LARIAT structure
poly(A) binding protein II (PABPII) binds to
the initial short poly(A) tail
ApoB-48
~240 kDa expressed in intestinal epithelia only one domain N-term: binds to lipids C is chemically turned into U which turns into stop codon
ApoB-100
~500 kDa, expressed in hepatocytes has two domains N-term: binds to lipids C-term: binds to LDL (for cholesterol transport)