Post-Transcriptional Events;
Other RNA Processing Events,
RNA editing
Introduction
Types of RNAs
Function
mRNA - messenger
Template for protein synthesis.
rRNA - ribosomal
Component of ribosome's (protein synthesis)
t-RNA - transfer
Transfer of amino acid (protein synthesis)
hnRNA - heterogeneous nuclear
Precursors & intermediates of mature mRNAs & other RNAs
(Premature mRNA)
scRNA - small cytoplasmic
Signal Recognition Particle (SRP)
tRNA processing
snRNA - small nuclear
snoRNA - small nucleolar
Participate in the splicing and transfer of hnRNA.
rRNA processing/maturation/methylation
miRNA-micro RNA
Usually endogenous, induce degradation of targeted mRNA. that block
expression of complementary mRNAs.
Regulation of transcription and translation
siRNA-small interfering RNA
Usually exogenous, induce degradation of targeted mRNA.
Regulation of transcription and translation
ncRNA-non-coding RNA
(npcRNA, nmRNA, fRNA)
all RNA other than mRNA,functional RNA molecule that is not
translated into a protein. longer than 200nt
Introduction

In a few organisms, other specialized pre-mRNA processing events occur, such
as trans-splicing

Most organisms process their rRNAs and tRNAs by more conventional
mechanisms

Eukaryotes control some of their gene expression by regulating
posttranscriptional processes, primarily mRNA degradation
Outline
1. Processing of rRNA (eukaryotic and
prokaryotic)
2. Processing of tRNA
3. Processing of sn-RNA
4. Trans-splicing
5. RNA editing
Ribosomal RNA (rRNA)
 In cell >80% of rRNA
 Serves to release mRNA from DNA
 Act as ribozymes in protein synthesis
 Relatively G:::C rich
 Ribosome
– Prokaryotes – 70S (50S & 30S)
• In 50S subunits - 23S & 5S
:31 proteins
• In 30S subunits - 16S
:21 proteins
– Eukaryotes – 80S (60S & 40S)
• In 60S sub-units – 28S, 5.8S and 5S
:50 proteins
• In 40S sub-units – 18S
:33 proteins
Ribosomal RNA Processing

Ribosomal RNAs are the most abundant and universal noncoding RNAs in
living organisms

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

No splicing occurs, only cutting (except Tetrahymena)
Eukaryotic

rRNA processing
Prokaryotic
Eukaryotic rRNA Processing

Ribosomal RNAs are made by pol I in eukaryotic nucleoli as precursors that
must be processed to release mature rRNAs

Processing uses Small Nucleolar RNA (snoRNA)

One of the medium-sized RNA which is approximately 60 to 170 nt

Primarily guide chemical modifications of other RNAs

Eukaryotic ribosomes have four distinct ribosomal RNAs.

In humans, the large subunits contains a 28S (in all eukaryote),5.8S and 5S
RNA molecule and small subunit contains an 18S RNA molecule.


Out of these three rRNA are carved by various nucleases from a single primary
transcript(pre-rRNA).

The 5S rRNA is synthesized from a separate RNA precursor outside the nucleolus.
Exact sizes of the mature rRNAs vary from one species to another
Eukaryotic rRNA Processing

Gene repeat, cluster;
nucleolus

Non-transcribed spacer
(NTS)

Transcribed spacers

Oscar Miller et al.; newt
nucleolus, Christmas tree
rRNA Processing (Eukaryotic Cell)
1.
5’-end of 45S precursor RNA is removed to 41S
2.
41S precursor is cut into 2 parts:
3.

20S precursor of 18S

32S precursor of 5.8S and 28S rRNA
3’-end of 20S precursor removed, yielding
mature 18S rRNA
4.
32S precursor is cut to liberate 5.8S and 28S
rRNA
5.
5.8S and 28S rRNA associate by base-pairing
6.
DNA cistron for 5S rRNA is present outside
Nucleolar organizer
7.
Transcription requires RNA pol III + TFIIIA,
TFIIIB & TFIIIC
rRNA Processing (Eukaryotic Cell)
 Electron
microscopy of human rRNA processing
intermediates, P. Wellauer and I. Dawid (1973)
rRNA Processing (Eukaryotic Cell)


rRNA-processing steps are orchestrated by snoRNAs, associated with proteins in
small nucleolar ribonucleoproteins, (snoRNPs)

A quite a few of many hundreds of snoRNPs participate in rRNA processing by
modifying nucleotides within the rRNA precursor

Each snoRNA binds to the specific portion of pre-rRNA to form an RNA-RNA duplex.

Dictate either methylation or pseudouridylation
rRNA precursor contains about 110 2’-O-methyl groups and about 100
pseudouridines


Help define what regions of the precursor to remove and what regions to preserve
Methyl groups as signal for processing

Methylation at 2’OH

110 CH3-group in 45 S; all preserved in final products
Bacterial rRNA Processing

Multiple copies of genes for rRNAs

Bacterial rRNA precursors contain 3 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
Bacterial rRNA Processing

In Bacteria 16S,23S and 5S rRNAs arise from single 30S RNA
precursor of about 6,500 nucleotides.
Bacterial rRNA Processing
tRNA

Transfer RNA/ Soluble RNA/ supernatant
RNA/ Adaptor RNA

Smallest among RNAs (75-93 nucleotides)

Recognizes codon on mRNA

Shows high affinity to amino acids

Carry amino acids to the site of protein
synthesis

tRNA is transcribed by RNA polymerase III

tRNA genes also occur in repeated copies
throughout the genome, and may contain
introns.
Transfer RNA Processing

tRNAs made as long precursors in all cells


Nuclei of eukaryotes have precursors of
single tRNA


Processed by removing RNA at both ends
Made by pol III
Bacteria, precursor may contain one or
more tRNA molecules or even rRNA

RNase III cleaves out individuals
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
Flanking regions of the 3’-OH and 5’ phosphate ends are cleaved
by the endonuclease action of RNase D and RNase P respectively.

RNase D one of the seven exoribonucleases identified in E. coli (3'-5'
exoribonuclease)

Add the 3' CCA sequence to t-RNA in prokaryotic t-RNA processing
Forming Mature 5’-Ends

Maturation of the 5’-end of a bacterial or eukaryotic tRNA involves removing extra nucleotides
from the 5’-ends of pre-tRNA in one step by an endonucleolytic cleavage catalyzed by RNase P
(all that is needed to form mature 5’-ends)

RNase P is a ribozyme—an enzyme in which RNA rather than protein is responsible for catalytic
activity.


Two domain: Specificity domain and catalytic domain

Bacteria: 1RNA + 1 protein subunit

Eukaryotes: 1 RNA + many protein subunits (11 in human)

Catalytic RNA subunit called M1 RNA

Bacterial RNase P contains a single protein subunit of about 120 amino acid residues.

Spinach chloroplast RNase P appears to lack an RNA subunit
Requires divalent metal ions (like Mg2+) for its activity.

Endo-ribonuclease responsible for generating 5’ end of matured tRNA molecules.

Cleavage via nucleophilic attack on the phosphodiester bond leaving a 5’-phosphate and 3’hydroxyl at the cleavage site.
Forming Mature 3’-Ends

3’-end maturation is more complex than 5’-maturation

6 RNases contribute to final trimming:

RNase D, RNase BN, RNase T, RNase PH, RNase II,and polynucleotide phosphorylase
(PNPase)

RNase II and PNPase cooperate to remove the bulk of the 3’-trailer from pre-tRNA

RNases PH and RNase T remove last 2 nucleotides

RNase T is the major participant in removing very last nucleotide
Forming Mature 3’-Ends

Processing of the 3′ end of tRNAs involves addition of a CCA
terminus, the site of amino acid attachment.

The enzyme tRNA nucleotidyl transferase adds CCA to the 3' ends
of pre-tRNAs.


Virtually all tRNAs end in CCA, forms the amino acceptor stem

For most prokaryotic tRNA genes, the CCA is encoded at the 3' end of the gene.

No known eukaryotic tRNA gene encodes the CCA, but rather it is added
posttranscriptionally by the enzyme tRNA nucleotidyl transferase. This enzyme is present in
a wide variety of organisms, including bacteria, in the latter case presumably to add CCA
to damaged tRNAs.
Bases are also modified at specific positions. About 10-15% of the
bases are modified.
Processing of tRNA
1.
Removal of leader sequence & trailer
2. Excision of an intron
3. Replacement of nucleotide
– Replacement of U residues at the 3′ end of
pre-tRNA with a CCA sequence
4. Modification of certain bases:
– Addition of methyl and isopentenyl groups
to the heterocyclic ring of purine bases
– Methylation of the 2′-OH group in the
ribose of any residue; and conversion of
specific uridines to dihydrouridine(D),
pseudouridine(y)
tRNA Modifications
 Post-transcriptional chemical modifications is an
essential part of the maturation process required
to generate functional tRNA molecules
 Over 100 chemically distinct post-transcriptional
tRNA modifications, that include methylations,
hydroxylations, deaminations, acetylations,
isomerizations and etc.
 Modifications can be divided into two major
groups:
1. Affect the overall structure of the tRNA
2. Target the functional centers of the tRNA
 Average 10-15% of the total residues are modified
in total tRNA
Crecy-Lagard et al.Nucleic Acids Research, 2019
Hopper A & Nostramo R. Frontiers in Genetics.2019
Processing of sn-RNA

Small nuclear ribonucleic acid (snRNA), also commonly referred to as U-RNA, is
a class of small RNA molecules that are found within the nucleus
of eukaryotic cells.

Intronless ,Non-polyadenylated, non-coding transcripts that function in the
nucleoplasm.

snRNAs can be divided into two classes on the basis of common sequence features and
protein cofactors
– The Lsm-class snRNA genes (U6 and U6atac) are transcribed by Pol III using specialized external
Promoters.
•
Lsm-class snRNAs never leave the nucleus.
– Sm-class genes are transcribed by a RNA polymerase II (Pol II)
•
Sm-class snRNAsare exported from the nucleus for cytoplasmic maturation events
•
Processed by ribonucleases
•
2 O’methylation and conversion of uridine to psuedouridine being the most common modifications of nucleosides
•
Involved in splicing and associated with Spliceosome Complex
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

Alternatively, trans-splicing has exons that are not part of the same gene at all,
may not even be on the same chromosome

Trans-splicing in several organisms:

Parasitic and free-living worms (C. elegans)

First discovered in African trypanosomes, a disease(African Sleeping Sickness)causing parasitic protozoan

5’ end of mRNA not match gene sequence; extra 35 nt shared with other mRNAs – called the spliced
leader (SL)sequence

Spliced leader (SL) is encoded separately, and there about 200 copies in the genome .

SL primary transcript contains ~100 nt that resemble the 5’ end of a mRNA intron
Possible Models to Explain the Joining of
the SL to the Coding Region of a mRNA
Trans-Splicing Scheme


Branchpoint A within halfintron 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
Trans-Splicing

Trans-splicing is very widespread in some organisms

In C.elegans all or nearly all mRNAs are trans-spliced to a small
group of spliced leaders

More than 15% of these trans-spliced mRNAs are encoded in groups of two
to eight genes that can be considered a kind of operon

Such a group of genes resembles a prokaryotic operon in that they
belong to a transcription unit controlled by a single promoter

It differs from a true operon in that the primary transcript is
ultimately broken into pieces by trans-splicing, with each coding
region being supplied with its own leader

Trans-splicing makes such eukaryotic “operons” possible by
providing each of the internal coding regions with its own cap
RNA Editing

Definition: Any process, other than splicing, that results in a change in the sequence of a RNA
transcript such that it differs from the sequence of the DNA template.

Relatively rare

First considered a bizarre relic; now recognized as widespread.

RNA editing is a process in which information change at the level of mRNA. It is revealed by
situations in which the coding sequence in an RNA differs from the sequence of DNA from which it
was transcribed.

Editing events may include the insertion, deletion, and base substitution of nucleotides within the
edited RNA molecule.

RNA editing occurs in the cell nucleus and cytosol, as well as within mitochondria and plastids.

Also occurs in a few chloroplast genes of plants, and at least a few nuclear genes in mammals.

RNA editing has been reported in: Protozoa, plants and mammals, not yet fungi or prokaryotes;
nuclear, mitochondrial, chloroplast, and viral RNAs; mRNA, tRNA, rRNA
RNA Editing

Discovered in trypanosome mitochondria

Unusual mitochondria called kinetoplasts,
which contain two types of circular DNA linked
together into large networks, highly packed
DNA (20% of total)

25–50 identical maxicircles, 20–40 kb in size,
which contain the mitochondrial genes


Sequencing of genomic Mt DNA (Maxicircles)
revealed apparent pseudogenes:

Full of Stop codons

Deletions of important amino acids
10,000 1–3-kb minicircles, which have a role in
mitochondrial gene expression
RNA Editing

Benne and colleagues sequenced cytochrome oxidase (COX II) mRNA and did
not actually code for the mRNA, but was a pseudogen

Pseudogenes are a duplicate copy of a gene that has been mutated so it does not
function and is no longer used

The mRNAs of trypanosomatids are copied from incomplete genes called
cryptogenes

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
RNA Editing
 Two
general types:
 Base
modification (deaminase)

A to I double-stranded mechanism, seen in viruses, human genes.

C to U, U to C seen in chloroplasts, plant mitochondria, human genes.
 Insertion/deletion

U insertion/deletion, seen in kinetoplastid protozoa

Mono/di nucleotide insertion, seen in Physarum

Nucleotide replacement, seen in Acanthamoeba tRNAs
Some Genes Are Very Heavily Edited!
COXIII
Cytochrome oxidase
III From Trypanosoma
brucei
Lower case Us were
inserted by editing.
The deleted Ts (found
in the DNA) are
indicated in upper
case.
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
K. Stuart
L. Simpson
Guide RNA (gRNA)

In general, RNA editing mechanisms are based on protein or protein-RNA
complexes and require a “guide RNA” molecule, which, through base-pairing
with the target RNA, determines the editing site

Guide RNAs (gRNAs) direct editing

gRNAs are small (40-70 nt) and complementary to portions of the mRNA

Structural elements: anchor, informational part and Oligo(U)tail

Base-pairing of gRNA with unedited RNA gives mismatched regions, which are
recognized by the editing machinery

Machinery includes an Endonuclease, a Terminal Uridylyl Transferase (TUTase),and a
RNA ligase

Editing is directional, from 3’ to 5
Model For The Role of gRNA in Editing

Guide RNAs (gRNA) could direct
the insertion and deletion of UMPs
over a stretch of nucleotides in the
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
Guide RNA Editing

5’-end of the first gRNA hybridizes
to an unedited region at the 3’border of editing in 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
Mechanisms of U Deletion/insertion

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 (terminal uridyl
transferase)
Editing Is Catalyzed by a Multiprotein Complex

A complex has been purified from glycerol gradients that contains the four key
enzymeactivities: 20S editosome

Endonuclease: cleavage in vitro occurs at an unpaired nucleotide immediately upstream
of the gRNA-mRNA anchor duplex.

Exonuclease: exoUase removes non-base-paired U nucleotides after cleavage of deletion
editing sites

TUTase: In insertion editing, Us are added to the 3’ end of the 5’ pre-mRNA fragment by
a terminal uridyl transferase as specified by the gRNA.

RNA ligase: the natural editing ligase substrates are nicked dsRNAs that are completely
base-paired after the correct addition or removal of U nucleotides

Helicase: each gRNA must be displaced from the sequence that it creates to enable
binding by the subsequent gRNA and also from the mRNA completely before translation

Other 20S editosome proteins
Other Systems with RNA Editing
1.
Land plant (C  U) and Physarum (slime mold)
mitochondria (nt insertions)
2.
Chloroplasts of angiosperms (C  U)
3.
Some nuclear genes in mammals
–
Apolipoprotein B, C  U
–
Glutamate receptor B, A  I (inosine)
4.
Hepatitus delta virus (A  I)
5.
Paramyxovirus (G insertions)
Editing by Nucleotide Deamination
1.
Some adenosines in mRNAs of higher eukaryotes, including fruit flies and mammals,
must be deaminated to inosine posttranscriptionally for mRNA to code for proper
proteins




Inosine largely behaves like a guanosine in RNA folding and is also interpreted as G by the
translation machinery
Enzymes know as adenosine deaminases active on RNAs (ADARs) carry out this kind of
RNA editing
Humans and mice contain three ADAR genes:
ADAR1, ADAR2, and ADAR3

ADAR1 & ADAR2 proteins are ubiquitous in the body

ADAR3 product is found only in the brain.

These enzymes are very specific
Drosophila genome contains only one ADAR gene (ortholog
ADAR2)
A to I Editing in RNA

Glutamate Receptor B

I read as G during
translation, R instead of Q

Affects Ca2+ permeability,
intracellular trafficking of
receptor

A-to-I RNA editing sites
also abundantly occur in
Intronic regions as well as
in 3′-UTRs
Editing by Nucleotide Deamination
2. Some cytidines must be deaminated to uridine for an mRNA to
code properly

Enzymes know as cytidine deaminase acting on RNA (CDAR) carry
out this kind of RNA editing
 C→U editing is defective in about 25% of the benign peripheral nerve sheath
tumors found in neurofbromatosis type I patients
 An example of C-to-U editing is with the apolipoprotein B gene in humans.
– Apo B100 is expressed in the liver and apo B48 is expressed in the intestines.
– The B100 form has a CAA sequence that is edited to UAA, a stop codon, in the
intestines. It is unedited in the liver.
Significance of RNA Editing

It is essential in regulating gene expression of organisms.

RNA editing mutant was reported with strong defects in
organelle development.

Deficiency causes diseases.

It is a mechanism to increase the number of different
proteins available without the need to increase the
number of genes in the genome.

May help protect the genome against some viruses.