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)