Transcript Other RNA Processing Events

Other RNA Processing Events
Chapter 16
Ribosomal RNA Processing
• 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 and each must
be cut out
Eukaryotic rRNA processing
• The rRNA are separated by regions called
nontranscribed spacers (NTSs).
• Transcribed spacers - regions of the gene that
are transcribed as part of rRNA precursor
- and then removed in processing of precursor
to mature rRNA species.
Eukaryotic rRNA Processing
• Ribosomal RNAs are made in eukaryotic
nucleoli as precursors that must be processed
to release mature rRNAs
• Order of RNAs in the precursor is (fig: 16.2)
–
–
–
–
18S
5.8S
28S in all eukaryotes
Exact sizes of the mature rRNAs vary from one
species to another
Eukaryotic rRNA Processing
• rRNA processing –
nucleolus – small nucleolar
RNAs (snoRNAs) +
proteins = Small nucleolar
ribonucleoproteins
(snoRNPs)
• E1, E2 and E3 – interact
with distinct regions in prerRNA
Bacterial rRNA Processing
• Bacterial rRNA precursors contain tRNA
and all 3 rRNA
• rRNA are released from their precursors by
RNase III and RNase E
– RNase III is the enzyme that performs at least the
initial cleavages that separate the individual
large rRNAs
– RNase E is another ribonuclease that is
responsible for removing the 5S rRNA from the
precursor
Processing Bacterial rRNA
Transfer RNA Processing
• Transfer RNAs are made in all cells as overly
long precursors
– These must be processed by removing RNA at
both ends
• Nuclei of eukaryotes contain precursors of a
single tRNA
• In bacteria - precursor may contain one or
more tRNA
- Sometimes a mixture of rRNAs and tRNAs
Cutting apart Polycistronic
Precursors
• In processing bacterial RNA that contain more
than one tRNA
– First step is to cut precursor up into fragments with
just one tRNA each
• Cutting between tRNAs in precursors having 2 or more
tRNA
• Cutting between tRNAs and rRNAs in precursors
– Enzyme that performs both chores is the RNase III
Forming Mature 5’-Ends of tRNA
• Extra nucleotides are
removed from the 5’ends of pre-tRNA in
one step by an
endonucleolytic
cleavage catalyzed by
RNase P
• RNase P from bacteria
and eukaryotic nuclei
have a catalytic RNA
subunit called M1 RNA
Processing 3’-Ends of tRNA
RNase D, RNase BN, RNase T, RNase PH, RNase II and polynucleotide
phosphorylase (PNPase)
Forming mature 3’-Ends of tRNA
• RNase II and polynucleotide phosphorylase
cooperate
– To remove most of extra nucleotides at the end of
a tRNA precursor
• RNases PH and T are most active in
removing the last 2 nucleotides from RNA
– RNase T is the major participant in removing very
last nucleotide
Trans-Splicing
• Splicing that occurs in all eukaryotic species
is called cis-splicing because it involves 2 or
more exons that exist together in the same
gene
• trans-splicing has exons that are not part of
the same gene at all - may not even be on
the same chromosome
The Mechanism of Trans-Splicing
• Trans-splicing occurs in several organisms
– Parasitic and free-living worms
– First discovered in trypanosomes
• Trypanosome mRNA are formed by transsplicing between a short leader exon and any
one of many independent coding exons
Trans-Splicing
Trans-Splicing Scheme
• Branchpoint adenosine
within the half-intron
attached to the coding
exon attacks the junction
between the leader exon
and its half-intron
• Creates a Y-shaped
intron-exon intermediate
analogous to the lariat
intermediate
Mechanism of Editing
• Unedited transcripts can be found along with
edited versions of the same mRNAs
• Editing occurs in the poly(A) tails of mRNAs
that are added posttranscriptionally
• Partially edited transcripts have been isolated always edited at their 3’-ends but not at their
5’-ends
Role of gRNA in Editing
• Guide RNAs (gRNA)
could direct the
insertion and deletion of
UMPs over a stretch of
nucleotides in the
mRNA
• When editing is done,
gRNA could hybridize
near the 5’-end of newly
edited region
Guide RNA Editing
• 5’-end of the first gRNA hybridizes to an
unedited region at the 3’-border of editing I the
pre-mRNA
• The 5’-ends of the rest of the gRNAs hybridize
to edited regions progressively closer to the 5’end of the region to be edited in the premRNA
• All of these gRNAs provide A’s and G’s as
templates for the incorporation of U’s missing
from the mRNA
Mechanism of Removing U’s
• Sometimes the gRNA is missing an A or G to pair
with a U in the mRNA
– In this case the U is removed
• Mechanism of removing U’s involves
– Cutting pre-mRNA just beyond U to be removed
– Removal of U by exonuclease
– Ligating the two pieces of pre-mRNA together
• Mechanism of adding U’s uses same first and last
step
• Middle step involves addition of one or more U’s
from UTP by TUTase
RNA Interference
• RNA interference occurs when a cell encounters
dsRNA from:
– Virus
– Transposon
– Transgene
• Trigger dsRNA is degraded into 21-23 nt fragments
(siRNAs) by an RNase III-like enzyme called Dicer
• Double-stranded siRNA, with Dicer and Dicerassociated protein R2D2 form a complex called
complex B
Complex B
• Complex B delivers the siRNA to the RISC loading
complex (RLC)
– Separates 2 strands of siRNA
– Transfers guide strand to RNA-induced silencing complex
(RISC) that introduces a protein- Ago2
Complex B
• The guide strand of siRNA base-pairs with target
mRNA in the active site of PIWI domain of Ago2
– Ago2 is an RNase H-like enzyme known as a slicer
– Slicer cleaves the target mRNA in middle of the region of
its base-pairing with the siRNA
– ATP-dependent step has cleaved RNA ejected from RISC
which then accepts a new molecule of mRNA for
degradation
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