BE 359 Ch. 10 Post-Transcriptional Gene Control

RNA

lampbrush chromosome, hnRNP protein associated with nascent _________ transcripts stained red with a monoclonal antibody

pre mRNA

______-_________ is capped, polyadenylated, spliced, and associated with RNPs in the nucleus before export to the cytoplasm

ribonucleoprotein spliceosome

A large _______________ _____________ complex catalyzes two transesterification reactions that join two exons and remove the intron as a lariat structure

splice sites

a network of interactions between SR proteins, snRNPs, and splicing factors forms a cross-exon recognition complex that specifies correct ________ _________

transcriptional regulation, mRNA processing, cytoplasmic regulation

gene expression regulation

RNA polymerase II

RNA processing:
RNAs from protein-coding genes -
transcribed by ________ ______________ ______

cytoplasm

RNA processing: RNAs from protein-coding genes -processed from primary transcripts in the nucleus before export to the ___________

alternative exons

RNA processing, step 1: use of ____________ ________ during pre-mRNA splicing

poly(A)

RNA processing step 2: use of alternative ____________ sites

exported

RNA processing, step 3:
properly processed mRNAs - _____________ to the cytoplasm

blocked

RNA processing, step 3: improperly processed mRNAs -
___________ from export to the cytoplasm

degraded

RNA processing, step 3: improperly processed mRNAs - ____________ in the exosome complex containing multiple ribonucleases

translation

RNA processing, step 4: translation initiation factors - bind to the 5′ cap cooperatively with poly(A)-binding protein I bound to the poly(A) tail and initiate ____________

mRNA degraded

RNA processing, step 5: ________ _______________ in cytoplasmic P bodies - translational repression,
deadenylated and decapped by enzymes

exosomes

RNA processing, step 5: degraded by cytoplasmic _____________

protein translated

RNA processing, step 5: of mRNA degradation rate - regulates mRNA abundance and amount of __________ ______________

polyA polymerase

RNA processing, step 6 : mRNAs synthesized without long poly(A) tails - translation regulated by controlled synthesis of a long poly(A) tail by a cytoplasmic ___________ ______________

inhibit translation

RNA processing, step 7 : translation regulation by other mechanisms--miRNA (~22-nucleotide RNAs) - ________ _____________ of mRNAs to which they hybridize, usually in the 3′ untranslated region

tRNAs

RNA processing, step 8a: __________- transcribed by Pol III and processed in the nucleus

rRNAs

RNA processing, step 8b: __________ - transcribed by Pol I - processed in the nucleolus

mature RNAs

RNA processing, step 9: regions of precursors cleaved from the __________ _______ - degraded by nuclear exosomes

transcription, 5' capping

cleavage at polyA site

polyadenylation

RNA splicing

mRNA

overview of __________ processing in eukaryotes

RNA polymerase II

eukaryotic cells convert an initial ________ ____________ ____ primary transcript into a functional mRNA by three modifications

5' capping, 3' cleavage and polyadenylation, RNA splicing

Eukaryotic cells convert an initial RNA polymerase II primary transcript into a functional mRNA by three modifications

initiates transcription

mRNA processing, step 1: nascent RNA (β-globin RNA) 5′ end capped with 7-methylguanylate (shortly after RNA polymerase II __________ ____________ at the first nucleotide of the first exon of a gene)

termination sites

mRNA processing, step 1: pol II transcription - terminates at any one of multiple ____________ ________ downstream from the poly(A) site in final exon

primary transcript

mRNA processing, step 2: cleavage enzyme cleaves __________ ___________ at the poly(A) site

polyadenylation

mRNA processig, step 3: ________________ enzyme adds a string of adenosine (A) residues (~250 A residues in mammals, ~150 in insects, and ~100 in yeast)

cleavage

mRNA processing, step 4:
short primary transcripts with few introns - splicing follows __________ and polyadenylation (shown)

nascent

mRNA processing, step 4: long transcripts with multiple introns--introns spliced out of the _________ RNA during transcription

mRNA

fully processed messenger RNA with 5' cap, introns removed RNA splicing, and a poly(A) tail

pre-mRNA

an mRNA precursor containing introns and not cleaved at the poly(A) site

hnRNA

heterogeneous nuclear RNAs, these RNAs include pre-mRNAs and RNA-processing intermediates containing one or more introns

snRNA

five small nuclear RNAs that function in the removal of introns from pre-mRNAs by RNA splicing, plus two small nuclear RNAs that substitute for the first two at rare introns

pre-tRNA

a tRNA precursor containing additional transcribed bases at the 5' and 3' ends compared with the mature tRNA, some pre-tRNAs also contain an intron in the anticodon loop

pre-rRNA

the precursor to mature 18S, 5.8S, and 28S ribosomal RNAs, the mature rRNAs are processed from this long precursor RNA molecule by cleavage, removal of bases from the ends of the cleaved products, and modification of specific bases

snoRNA

small nucleolar RNAs, these RNAs baes-pair with complementary regions of the pre-rRNA molecule, directing cleavage of the RNA chain and modification of bases during maturation of the rRNAs

siRNA

short interfering RNAs, around 22 bases long, that are each perfectly complementary to a sequence in an mRNA, together with associated proteins, siRNAs cause cleavage of the "target" RNA, leading to its rapid degradation

miRNA

micro-RNAs, around 22 bases long, that base-pair extensively, but not completely, with mRNAs, especially over bases 2 to 7 at the 5' end of the miRNA (the "seed" sequence), this pairing inhibits translation of the "target" mRNA and targets it for degradation

premRNA splicing

two transesterification reactions at short, conserved sequences

splice

Consensus sequences around _______ sites in vertebrate pre-mRNAs

flanking bases

Intron splice site invariant bases (____________ _______ indicated - found at frequencies higher than expected for a random distribution)

branch-point

usually 20-50 bases from the 3′ splice site

GU, AG

intron splice site invariant bases: 5′ _______, 3′ ______, branch-point adenosine

polypyrimidine tract

_________________ ______ near the 3′ end of the intron - found in most introns

normal

40 bases-50 kilobases, only 30-40 nucleotides at each end of an intron are necessary for splicing to occur at ___________ rates

exon splicing

two sequential transesterification reactions [Arrows indicate where activated hydroxyl oxygens react with phosphorus atoms]

splicing

two transesterification reactions result in the _____________ of exons in pre-mRNA

reaction 1

two transesterification reactions: ____________ _____- intron 5′ phosphorus-exon one 3′ oxygen ester bond - exchanged for an intron 5′ phosphorus ester bond with the branch-point A residue 2′ oxygen

reaction 2

two transesterification: ___________ _____ -the exon two 5′ phosphorus - intron 3′ oxygen ester bond - exchanged for an exon two 5′ phosphorus ester bond with the 3′ oxygen of exon one

joins

two transesterification: reaction 2--_______ the two exons
releases intron as a lariat structure

base pairing

________ __________ between pre-mRNA, U1 snRNA, and U2 snRNA early in the splicing process

pre mRNA

snRNAs involved in splicing: five U-rich snRNAs - U1, U2, U4, U5, and U6 (107-210 nucleotides long) - participate in ______-_____________ splicing

base pair

snRNAs involved in splicing: ________-__________ with pre-mRNA

nuclear ribonucleoprotein particles

snRNAs involved in splicing: interact with 6-10 proteins each - form small __________ _______________ ____________ (snRNPs)

U1, U2

________ and _______ base-pairing with pre-mRNA for splicing: (purple rectangles - sequences that bind snRNP proteins recognized by anti-Sm protein antibodies)

U1

U1 and U2 base-pairing with pre-mRNA for splicing: _______- base-pairs across 5' splice site exon-intron junction

U2 snRNA

U1 and U2 base-pairing with pre-mRNA for splicing: _______ __________-- base-pairs with sequence surrounding the branch-point A, unbase-paired branch-point A bulges out to allow 2'-OH to participate in first transesterification reaction

U1 snRNA, splicing

mutations that inhibit or restore splicing: (left) – mutation (A) in a pre-mRNA splice site, interferes with base pairing to the 5′ end of ______ ________, blocks ___________

base pairing

mutations that inhibit or restore splicing: (right) – U1 snRNA with a compensating mutation (U) – restores ________ ____________, restores splicing of the mutant pre-mRNA

spliceosome

model of __________-mediated splicing of pre-mRNA

five U-rich snRNAs

snRNAs involved in splicing: U1, U2, U4, U5, and U6 (107–210 nucleotides long),

participate in pre-mRNA splicing

proteins

snRNAs involved in splicing: five U-rich sRNAs, base pair with pre-mRNA, interact with 6-10 __________ each

small nuclear ribonucleoprotein particles

snRNAs involved in splicing: interact with 6-10 proteins each--form _______ ____________ ______________ _______ (snRNPs)

spliceosome

five splicing snRNPs and splicing proteins assembled on a pre-mRNA

U1

spliceosome splicing mechanism, initial complex assembly:
_____ - base-pairs with the consensus 5′ splice site

splitting factor 1

spliceosome splicing mechanism, initial complex assembly: ____________ ____________ ____ (SF1) binds the branch-point A

U2 snRNP associated factor

spliceosome splicing mechanism, initial complex assembly: ______ __________ ____________ ___________ (U2AF) associates with the polypyrimidine tract and 3′ splice site

U2 snRNP

spliceosome splicing mechanism, step 1: _____ _________ - associates with the branch-point A via base-pairing interactions and displaces SF1

spliceosome

spliceosome splicing mechanism, step 2: U4-U5-U6 trimeric snRNP complex - binds to form the ______________

active

spliceosome splicing mechanism, step 3: snRNA base-pairing interaction rearrangements--
convert the spliceosome into a catalytically _________ conformation

U1, U4

spliceosome splicing mechanism, step 3: snRNA base-pairing interaction rearrangements--_______ and ______ snRNPs - released

transesterification

spliceosome splicing mechanism, step 4: First _____________ reaction - U6-U2 catalytic core catalyzes formation of intermediate containing a 2′,5′-phosphodiester bond

phosphodiester

spliceosome splicing mechanism, step 5: second transesterification reaction--joins the two exons by a standard 3′,5′-_____________ bond

intron

spliceosome splicing mechanism, step 5: second transesterification reaction--releases the __________ as a lariat structure

releases

spliceosome splicing mechanism, step 5: second transesterification reaction--__________ remaining snRNPs

linear RNA

spliceosome splicing mechanism, step 6: debranching enzyme coverts excised lariat intron into a __________ _______for degradation

cooperative binding

exon recognition through _____________ ___________ of SR proteins and splicing factors to pre-mRNA

sr proteins

named because they contain a protein domain with long repeats of serine and arginine amino acid residues, whose standard abbreviations are "S" and "R" respectively

sr proteins

interact with exonic enhancer sequences (ESEs) within exons

sr proteins

contribute to exon definition in long pre-mRNAs

exons

pre-mRNAs humans: avg ~150 bases

introns

pre-mRNAs humans: avg ~3500 bases - longest exceed 500 kb

degenerate

pre-mRNAs humans: _____________ 5′ and 3′ splice site and branch point sequences - multiple copies likely to occur randomly in long introns

spliced together

pre-mRNAs humans: additional sequence information is required to define the exons that should be _________ ______________ in higher organism pre-mRNAs with long introns

arginine, serine

SR proteins: contain several RS protein-protein interaction domains rich in ___________ (R) and ___________ (S) residues

exonic enhancer sequences

SR proteins:
interact with _________ ___________ ____________ (ESEs) within exons

protein protein

SR proteins: mediate cooperative binding of U1 snRNP to a true 5′ splice site and U2 snRNP to a branch point through a network of ___________-____________ interactions that span an exon

GU, AG

5' _______ and 3' _________ splice sites recognized by splicing factors on the basis of their proximity to exons

interact

SR proteins bound to ESEs: ________ with each other

downstream

SR proteins bound to ESEs: promote cooperative binding of--U1 snRNP to the 5′ splice site of the ______________ intron

upstream

SR proteins bound to ESEs: promote cooperative binding of--U2 snRNP binding to the branch point of the upstream intron, the 65- and 35-kDa subunits of U2AF to the polypyrimidine tract and AG 3′ splice site of the ____________ intron, and other splicing factors

protein cross exon

resulting RNA-________ ____________-_________ recognition complex - spans an exon and activates the correct splice sites for RNA splicing

spinal muscular atrophy

one of the most common genetic causes of childhood mortality

identical

SMN1 and SMN2 genes--encode ___________ proteins

silent

SMN2 ____________ mutation interferes with SR protein binding