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Chapter 8

Various RNA definitions (pre-mRNA, hnRNA, snRNA, snoRNA, miRNA, siRNA)


Pre-mRNA: the precursor to mature 18S, 5.8S, and 28S ribosomal
RNAs. The mature rRNAs of these are processed from this long
precursor RNA molecule by cleave, removal of bases from the ends of
the cleaved products, and modification of specific bases
hnRNA: Heterogenerous nuclear RNAs include pre-mRNA and RNA
processing intermediates containing one or more introns
snRNA: these are five small nuclear RNAs that remove introns from pre-
mRNAs by RNA splicing, plus two small nuclear RNAs that substitute for
the first two at rare intron regions.
snoRNA: are small nucleolar RNAs that can base-pair with
complementary regions of the the pre-RNA molecule which directs
cleavage of the RNA chain and modifies bases during maturation of
rRNAs
miRNA: are micro RNAs that are 22 bp long, they base-pair extensively
and incompletely with mRNAs at the 6 bp at the 5' end of the miRNA in
order to hibit translation of the "target" mRNA
siRNA: are short interfering RNAs, that are 22 bp long that complement
to a sequence in mRNA. Once attached to a protein, siRNA cleaves off
the "target"RNA, to its degradation.

Capping reactions, order and enzymatic activities involved


Capping reactions are done for the RNA to avoid its degradation during
translocation. Only 10% of pre-mRNA gets capped.
Steps: Capping Reaction
-1. Phosphohydrolase:
-2. Guanylyl Transferase:
-3.Guanine-7-Methyl Transferase:
-4. 2'-O-Methyl Transferase:
*capping enzymes (above) are associated with the
phophorylated CTD of pol II once it initiates synthesis
(capping~elongation)

hnRNPs (heterogeneous nuclear RNPs) and hnRNP protein functions


hnRNPs are heterogenous nuclear RNPs that are associated with
mRNAs during synthesis in the nucleus. They are required for
processing and also serve to block the formation of pre-mRNA
secondary structures
They can usually be found hanging out at The Nucleus and The
Cytoplasm
They are portray RNA-binding motifs
GU and AG conserved sequences at ends of introns
GU and AG are invariant at the ends of introns which makes splicing
easier to do, the rest of the introns can be variable if they want to for
the reason that only 30-40 nt of invariant sequences at each end are
necessary for optimal splicing.

A residue branch point in intron and pyrimidine-


rich region (approximate distance from intron 3
end)

Transesterification reactions in splicing (1st and


2nd)
Transesterification is the exchange of a
phosphate ester bond with another
Steps: Transesterification
-1. 1st reaction cuts at the 5' site
and links a 5' phosphate of an intron to a
2' OH of residue A at the branch
site.
-2. 2nd reaction cuts at 3' site and
links a 2' OH (from the exon upstream)
to the phosphate (from exon's
downstream)

Intron lariat structure and 2-5 bond


An Intron lariat that has been excised is
debranched by an enzyme and then
degraded

snRNAs and snRNPs (U1, U2, U4, U5, U6)


snRNAs (107-210 bp long) are required for
pre-mRNA splicing.
The aggregation of these snRNAs of individual U1, U2, U4, U5, U6), the
five total will attract 6- 10 proteins to form snRNPs. U1 and U2 base
pair with consensus sequences at splice junctions.
snRNPs are ribonucleo protein particles

U1 and U2 base-pairing to pre-mRNA, compensatory mutation evidence, and


spliceosome assembly (5)
U1 base pairs to the 5' site and U2 base pairs to the 3' site
Compensatory mutation evidence: mutations that disrupted base pairs
were then base paired by a compensatory mutation to restore base
pairing
Spliceosome assembly:
-After U1 base pairs at the 5' site, SF1, binds to "branch point" (right
next to it) and U2AF binds to the Yn track (next to SF1).
-After U2 binds to the 3' end, followed by the attachement of trimeric
snRNP (U4,U5,U6) in order to form a spliceosome.
-First Transesterification is the release of U1 and U4 and can occur if
base pairing is rearranged within the spliceosome to make for a
catalytically active conformation.
-Second Transesterification is caused by a second rearrangement
within the spliceosome and release of the remaining snRNPs
*spliceosomes contain spliceosome factors (proteins)

Phosphorylated CTD structure and role in recruiting mRNA precursor


processing enzymes
Pol II CTD couples pre-mRNA processing with transcription elongation
by allowing multiple proteins to be associated with a single transcribing
polymerase. This can only be done with capping enzymes,
polyadenylation factors, and splicing factors.
*the higher the concentration of these proteins, the greater the
rate and specificity of processing splice sites and poly (A) signals

Exonic splicing enhancer sequences and SR proteins (6)


Exionic Splicing Enhancers (ESE) are flanked exon sequences that
enables splicing site recognition and the conserved consensus
sequence at both 5' and 3' ends of an intron ensure a splicing reaction
SR proteins (contain amino acid residues S and R) bind to sites on the
ESE, U1 snRNP to form a cross-exon recognition complex that allow
precise specificity of splice junctions and regulate alternative splicing

Self-splicing introns and where they are found in nature


In rare instances, introns can self-splice themselves out of the pre-
cursor RNA if there is an absence of proteins
This phenomena can occur in rRNA in protozoans and in tRNA in
mitochondria and chloroplasts. In vivo reactions can be aided by
maturases.

AAUAAA polyadenylation signal and CPSF (cleavage and polyadenylation


specificity factor)
The sequence AAUAAA and a G/U rich sequence in the pre-mRNA
allows for polyadenylation. A CPSF (cleavage and polyadenylation
specificity factor)protein will bind to the AAUAA sequence like a landing
dock and a CStF (Cleavage stimulatory factor) complex will bind to the
G/U sequence the same.
*looped RNA stabilizes its complex by binding to cleave factors:
CFI and CFII. Poly (A) polymerase (PAP) is attracted to the
complex to activate the CPSF-mediated cleave downstream of the
AAUAAA sequence

Coupled cleavage and polyadenylation (9)


Steps
-1. After cleavage is completed (polyadenylation), CStF, CF1, and
CF2 are released and PAP adds 12 amino acid residues at a slow rate.
-2. PABPII is then binded to a short poly (A) tail and accelerates
the addition of A residues by PAP

PAP (poly(A) polymerase) and two phases of poly (A) addition


Phase 1: PAP adds 12 "A" residues at a slow rate
Phase 2: PABPII binds to a short poly (A) tail to accelerate the addition
fo A residues by PAP

PABP II (poly(A)-binding protein II)


PABP II is used in phase II of polyadenylation to cleave the pre-mRNA
and is required in a large amount to the growing tail. It will stop once it
reaches 20-250 "A" residues added
*exosomes is a large nuclear complex of exonucleases that
degrade regions downstream of cleavage sites on pre-mRNA.

Regulated alternative splicing in Drosophila sex determination (role of Sxl,


Tra, and Dsx proteins) (10)
In order to regulate alternative splicing proteins that promote splicing
or promote skipping of specific exons are required.
Specific proteins:
-1. Sxl: regulates alternative splicing (which thereby produces Dsx
protein) and promotes skipping of an intron
-2. Tra: is a transformer protein which promotes joining of two exons.
-3. Dsx: this is produced in two isoforms, one is male-specific
repression of transcription of female-specific genes. The other is
female-specific repression of male-specific genes.

Dscam gene and general role of alternative splicing in neuron synaptic


connections (11)
Dscam genes regulates synaptic connections between neurons that
can produce 38K isoforms. Alternative splicing is important in neuron
synaptic connections because it modulates neural function.

K+ channels in hair cells and role of alternative splicing


K+ channels in hair cells are alternatively spliced mRNAs that activate
in response to Ca 2+. Different isoforms of the K+ channel are
activated depending on the specific [Ca 2+]

RNA editing in apolipoprotein B gene


RNA editing, common in mitochondria, alters the coding sequence of a
mRNA after it has been synthesized by changing a single base.
RNA editing occurs in apolipoprotein B gene which is part of the LDL
complex that delivers chelesterol and is a crucial role in
atherosclerosis.

Nuclear pore complex, nucleoporins and FG-nucleoporins (what does FG


stand for?)
Nuclear pore complex: a complex that spans both layers of a nuclear
membrane, that is the gateway to all transport throughout the
nucleus. It has 8 filaments extending on either side.
nucleoporins: are proteins that line the interior walls of the central
channel of the NPC
FG-nucleoporins: they also line the central channel and are rich in "F"
and "G" residues
*larger complexes require a transporter protein to cross the
nuclear envelope. mRNPs are exported via exportins or importins.
Exosomes degrade improper mRNPs before translocation.

NXF1/NXT1 transporter protein for mRNP and mode of interaction with FG-
nucleoporins
NXF1/NXT1 is a heterodimer that binds to parts of teh exon-junction
complexes in order to export mRNP.
hydrophobic regions of this heterodimer can reversibly bind to the FG-
nucleoporin domain and aid in mRNP diffusion through the NPC central
channel

mRNP remodeling (CBC [nuclear cap-binding complex], and poly(A) binding


proteins)
mRNP is remodeled during nuclear export by its associated protein
degradation before/after transport.
mRNP remodeling is when mRNPs scout for new proteins after reaching
its destination cytoplasm. Phosphorylation of some mRNP proteins will
also take place for mRNP export.
During remodeling
* Nuclear cap-binding complex (CBC) is replaced by eIF4E
* PABPII is replaced by PABP1
eIF4E (cytoplasmic cap-binding protein, subunit of eIF4 translation initiation
factor)

PABPI (cytoplasmic poly(A) binding protein I)


miRNAs (micro RNA) and siRNAs (silencing RNA), properties and mode of
action (14)
miRNAs are micro RNAs that involve imperfect-base-pairing and inhibit
target mRNA translation
siRNAs are short interference RNAs that involve perfect-base-pairing,
cause mRNA degradation, defend again virus, and block excessive DNA
transposition
BOTH are short-single stranded RNAs that base pair to target RNAs

miRNA processing (hairpin precursor, role of Drosha and Dicer)


miRNA base pairs to the 3' UTR of the target mRNA and regulate gene
expression/translation of many mRNAs with similar (imperfect bp-ing)
3' ends
hairpin precursors are primary miRNA transcripts that can encode
multiple miRNAs about 70 nt long. Its introns and 3' UTRs can also
process miRNA
Drosha is a dsRNA-specific nuclease and aids in producing the
precursor hairpin, pri-miRNAs, in the nucleus to then be exported out
of the nucleus
Dicer is a cytoplasmic dsRNA nuclease that produces pre-miRNAs and
small dsRNAs that are unpaired at its ends, with the aid of TRBP.

RISC (RNA-induced silencing complex), Argonaute subunit function


RISC (RNA-induced silencing complex) is the processed targeted
complementary strand to the mRNA and bind to the 3' UTR to inhibit
translation
Argonaute is a protein in the RISC complex that serves to inhibit mRNA
translation

cytoplasmic polyadenylation in fertilized eggs (CPE element, CPEB protein,


maskin, and role of phosphorylation in recruiting PAP and CPSF), role of eIF4G
and PABP in translation (covered in Chapter 4)
Cytoplasmic control element (CPE): is in mRNAs in its 3' UTR region
upstream from the poly (A) signal
Steps: Cytoplasmic Polyadenylation
-1. CPEB protein binds to CPE, then to the maskin protein, then to
the eIF4E (blocks normal interaction with the eIF4G).
-2.Fertilization causes CPEB phosphorylation and dissociation of
maskin of the complex
-3. CPSF (cleavage and polyadenylation specific factor) binds to a
poly (A) site and phosphorylates CPEB
-4. CPSF recruits cytoplasmic PAP to make long poly (A) tails,
PABPI binding, and restoration of translation.

Pathways of mRNA degradation (deadenylating enzyme, decapping enzyme,


P-bodies)
mRNA degradation is governed by specific sequences in their UTR
regions
deadenylating enzyme: this enzyme gradually shortens the poly (A),
decreases PABPI binding, destabilizes eIF4E's binding to cap, and
removes exposed cap
decapping enzyme: removes exposed cap.
P-bodies: are processing bodies that contain decapping enzymes and
one of the bidirectional (5' to 3') exonuclease. miRNA-RISC complexes
and UTR regions can be found here

*all pathways require the exosome complex (with bidirectional


exonucleases) in the final step of degradation

Exosome (complex of 3 to 5 and 5 to 3 exonucleases)


exosomes increase degradation

AU-rich sequences in 3 UTR of short-lived mRNAs


AU-rich sequences serve as binding sites for proteins that stimulate
deadenylating enzymes and the exosome

Iron-dependent regulation of ferritin and transferring receptor mRNA


translation (IRE, IRE-BP)
Ferritin is an iron binding protein that stores excess iron inside the cell
but is shut off when iron is low. Its 5' UTR contains 2-IREs (iron
response elements)
Transferrin is a protein that delivers iron from food to cells by TfR
(transferrin receptor) which is located on the cell's surface. It contains
an AU-rich region at its 3' UTR which promotes its own degradation
when [Fe] is high.
*3' UTR also contains 3-IREs and one overlaps the AU-rich region
Steps: Iron Dependent Translation of Ferritin
-1. IRE-BP (IRE-binding protein) binds to IREs when [Fe] is low and
blocks mRNA 5' scanning by the 40S ribosome subunit.
*high [Fe] promotes iron binding to IRE-BP which prevents
binding to IREs and allows ferritin protein translation
*IRE-BP binding to Tfr mRNA = promotes translation
IRE-BP binding to ferritin mRNA = blocks translation

Role of mTOR and eIF2 kinases in global translation


mTOR: is a protein kinase (target of the rapamycin antibiotic) and
serves in regulation. It can stimulate transcription and translation by
phosphorylating an inhibitor of eIF4E or S6 (a protein kinase that
phosphorylates ribosomal proteins).
It can also downregulate due to stressors by activation protein kinases
that phosphorylate eIF2 in order to block translation

Nonsense-mediated decay of mature mRNAs (pioneer round of translation,


exon-junction complexes, Upf1, P-bodies) (22)

mRNA localization signals in 3 UTR


rRNA processing (transcription unit, role of snoRNPs, methylation,


pseudouridylation)

Ribosome assembly (pre-90s, pre-60S)


tRNA processing (RNase P, origin of 3 CCA sequence, small intron in rare


tRNA precursors, base modifications)

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