Download Chapt 16: Other RNA Processing 16.1 Ribosomal RNA Processing

3/30/2011
Chapt 16: Other RNA Processing
Student learning outcomes:
• Explain how rRNA precursors are cleaved to give
final products
• Explain how tRNA precursors are trimmed, modified
• Describe how trans-splicing and RNA editing
occur in some protists or parasitic worms
• Describe how RNA interference (RNAi) uses ds
RNA to degrade specific mRNA
• Figures: 1, 2*, 3, 4*, 5*, 7, 10, 13, 14, 17, 20, 29, 31, 33*, 36*,
37*, 38, 40, 45
• Problems: 1, 2, 3, 5, 16, 17, 20, 22, 23; AQ 1
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16.1 Ribosomal RNA Processing
• mRNA in eukaryotes frequently requires splicing,
but does not undergo any trimming from ends
• rRNA genes of both eukaryotes and bacteria are
transcribed as larger precursors; must be
processed to yield rRNAs of mature size
• Several different rRNA molecules are embedded in
a long, precursor; each must be cut out
• No splicing occurs, only cutting
– (except Tetrahymena)
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Eukaryotic rRNA
Processing
• Ribosomal RNAs made by pol I
in nucleoli are precursors:
process to release mature
rRNAs
• Processing uses snoRNAs
(snoRNPs)
• Order of RNAs in precursor:
– 18S
– 5.8S
– 28S in all eukaryotes
Exact sizes of mature rRNAs
vary among species
Fig. 1 transcription by pol I
of Newt rRNA genes;
Many rRNA from 1 gene
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Processing of
rRNA in
Human Cells
1. 5’-end of 45S precursor RNA
is removed to 41S
2. 41S precursor is cut in 2:
–
–
20S precursor of 18S rRNA
32S precursor of 5.8S, 28S
3. 3’-end of 20S precursor
removed, yielding mature
18S rRNA
4. 32S precursor is cut to give
5.8S and 28S rRNA
5. 5.8S and 28S rRNA
associate by base-pairing
Fig. 2
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Bacterial rRNA Processing
• Multiple copies of genes for rRNAs
• rRNA precursors contain tRNA, the 3 rRNAs
• rRNAs are released by RNase III and RNase E:
– RNase III performs at least the initial cleavages that
separate individual large rRNAs
– RNase E removes 5S rRNA from precursor
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16.2 Transfer RNA Processing
• tRNAs made as long precursors in all cells
– processed by removing RNA at both ends
• Nuclei of eukaryotes have precursors of single tRNA
– Made by pol III
• Bacteria, precursor may contain one or more tRNA
molecules or even rRNA
– RNase III cleaves out individuals
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RNase P Forms Mature 5’-Ends of tRNA
• Extra nucleotides removed
from 5’-ends of pre-tRNA by
endonucleolytic cleavage
catalyzed by RNase P
• RNase P has catalytic RNA
subunit - M1 RNA
– (bacteria and eukaryotic nuclei)
Norm Pace, Sid Altman
• Catalysis requires Mg++
• RNase P also has protein
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Fig. 5
RNases Form Mature 3’-Ends of tRNA
• 6 RNases contribute to final trimming:
– Including RNase D, RNase BN
• RNase II and polynucleotide phosphorylase
(PNPase) remove most extra nucleotides to +2
• RNases PH and T remove last 2 nucleotides
Fig. 7
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16.3 Trans-Splicing
• Most splicing is cis-splicing: 2 or more exons from
same gene
• trans-splicing - exons not part of same gene; may
not even be on same chromosome
– Trypanosome mRNA: trans-splicing between leader exon
(splice leader, SL) and one of many independent exons
• Trans-splicing in several organisms
– Parasitic and free-living worms (C. elegans)
– First discovered in trypanosomes
(5’ end of mRNA not match gene sequence; extra 35 nt
shared with other mRNAs)
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Trans-Splicing Scheme
• Branchpoint A within
half-intron attached to
coding exon attacks
junction between
leader exon and its
half-intron
• Creates Y-shaped
intron-exon
intermediate
analogous to lariat
intermediate
Trypanosome and red
blood cell
Fig. 10
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16.4 RNA Editing
• Pseudogenes - duplicate
copies of genes, mutated, no
longer function or not used
• Cryptogenes - incomplete
genes
• Trypanosomatid mitochondria
cryptogenes for COX II encode
incomplete mRNA - must be
edited before translated
• Editing occurs 3’→
→5’ direction
by successive actions of guide
RNAs to insert/ delete Us
Fig. 13 minicircles regulate;
maxicircles are
mitochondrial genes
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Mechanism of Editing
• Unedited transcripts found with edited versions of
same mRNAs
• Editing occurs in poly(A) tails of mRNAs, that were
added posttranscriptionally
• Partially edited transcripts isolated, always edited at
their 3’-ends but not at 5’-ends
Fig. 14 example of edited section of Cox II
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Role of gRNA in Editing
• Guide RNAs (gRNA) could
direct insertion and deletion
of UMPs in mRNA
• 5’-end of gRNA hybridizes
to unedited region at 3’border of editing pre-mRNA
• When editing is done,
another gRNA could
hybridize near 5’-end of
newly edited region
Fig. 17
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RNA Editing
• gRNAs provide A’s and
G’s as templates for
incorporation of U’s
missing from mRNA;
• Some G-U bp are used
Figs. 18, 20; mechanisms
of insertion, deletion
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Mechanism of Removing, Adding U’s
• If gRNA is missing A or G to pair with U in mRNA
– Then U is removed
• Mechanism of removing U’s involves
– Cutting pre-mRNA just beyond U to be removed
– Removal of U by exonuclease
– Ligating two pieces of pre-mRNA together
• Adding U’s uses same first and last step
• Middle step involves addition of one or more U’s
from UTP by TUTase (terminal uridyl transferase)
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16.5 Posttranscriptional Control of Gene
Expression
• Common form of posttranscriptional control of gene
expression is control of mRNA stability
• Example: mammary gland tissue stimulated by
prolactin -> increase synthesis of casein protein
– Most increase in casein not due to increased rate of
transcription of the casein gene
– Is increase in half-life of casein mRNA
Example: transferrin receptor mRNA stability and
response to iron concentration
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RNA Interference:
posttranscriptional control
• RNAi - selective inhibition of expression of genes
• Originally antisense RNA thought to block
translation by binding mRNA; sense strand also
works, and dsRNA is best
Fig. 29 C. elegans.
Inject antisense or ds RNA to
mex-3; In situ hybridize
embryos to mex-3 probe.
a) Negative; No probe;
b) Positive hybridize, no RNAi
c) Antisense mex-3
d) ds RNA to mex-3
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RNA Interference
• RNA interference - cell encounters
dsRNA from:
– Virus, Transposon, ds RNA
• Trigger dsRNA degraded to 21-23 nt
fragments (siRNAs, short interfering
RNA) by Dicer, RNase III-like
• ds siRNA, with Dicer, Dicer-associated
protein R2D2 form Complex B
• Complex B delivers siRNA to RISC
loading complex (RLC)
– Separates 2 strands of siRNA
– Transfers guide strand to RNAinduced silencing complex (RISC) that
Fig. 33 Model
includes protein Argonaute2 (Ago2)
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• Guide strand of siRNA base-pairs
with target mRNA in active site of
PIWI domain of Ago2
– Ago2 is RNase H-like enzyme
known as Slicer
– Slicer cleaves target mRNA in
middle of region of base-pairing
with siRNA
– Ago2 purified from an Archaeaon
ATP-dependent step -> cleaved
RNA ejected from RISC,
RISC then accepts new molecule of
mRNA for degradation
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Specificity of RNAi, Complex B, RLC, RISC (Ago2)
Fig. 36 Ago2 + siRNAs for 2 sites; RNAi requires Mg++
Fig. 37 Assembly of the complexes
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Physiological role, usefulness of RNAi
Possible physiological role
• Fight ds viruses
• Prevent endogenous transposons
• Silence transgenes
Usefulness to experimenters:
• Tool to study basic principles –affect phenotype
• Potential to silence oncogenes
• ShRNAs provided long-lived research tool (short
hairpin RNAs)
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Amplification of siRNA
• siRNA is amplified during RNAi when antisense siRNAs
hybridize to target mRNA and prime synthesis of full-length
antisense RNA by RdRP (RNA-dependent RNA polymerase)
• New dsRNA is digested by Dicer into new pieces of siRNA
Fig. 38
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RNAi is involved in Heterochromatin
Formation in yeast
• Genes at yeast centromeres are heterochromatin
and silenced; also silent mating-type regions
• Yeast mutant in genes for Dicer, Ago and RdR are
defective in silencing genes near centromere
• Fission yeast Schizosaccharomyces pombe has
active transcription of reverse strand at outermost
regions of centromere
– Rare forward transcripts can base-pair with reverse
transcript to trigger RNAi
– Recruits histone methyltransferase, methylates Lys-9 of H3
– This recruits Swi6, causing heterochromatization
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RNAi is involved in Heterochromatin Formation in
fission yeast near centromere
Rare forward transcript -> ds RNA, Dicer, Ago1 and RITS
complex, methylation of histones, heterochromatin
Fig. 40
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Heterochromatin Formation in Plants and
Mammals
• Formation of heterochromatin aided by DNA
methylation
• methylation of C of CpG sequences attracts
heterochromatization machinery
• Individual genes silenced in mammals by RNAi that
targets gene’s control region rather than coding
region (ex. X-inactivation)
• Silencing involves DNA methylation rather than
mRNA destruction
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MicroRNAs (miRNAs) and Gene Silencing
• MicroRNAs - 18-25 nt RNAs produced by cleavage
from 75-nt stem-loop precursor RNA
• Dicer RNase cleaves ds stem part of precursor to
yield miRNA in ds form
• Single-stranded form of miRNAs joins Argonaute
protein in RISC to control gene expression by basepairing to mRNAs
– In animals, miRNAs tend to base-pair imperfectly to 3’UTRs of target mRNAs -> inhibition of protein product
accumulation of such mRNA
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Pathways to
Gene
Silencing by
miRNAs
Fig. 45
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Review questions
1. Draw structure of mammalian rRNA precursor, showing
locations of 3 mature rRNAs.
2. What is function of RNAseP? What is unusual about the
enzyme?
3. What is the difference between cis- and trans-splicing?
5. Describe RNA editing. What is a cryptogene?
16. Present a model for mechanism of RNA interference
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