Central dogma

The central dogma of molecular biology is a theory stating that genetic information flows only in
one direction, from DNA, to RNA, to protein, or RNA directly to protein.

1.) Replication: (DNA to DNA):

General Overview of a DNA Replication Fork. At the origin of replication, topoisomerase II
relaxes the supercoiled chromosome. Two replication forks are formed by the opening of the
double-stranded DNA at the origin, and helicase separates the DNA strands, which are coated
by single-stranded binding proteins to keep the strands separated. DNA replication occurs in
both directions.

An RNA primer complementary to the parental strand is synthesized by RNA primase and is
elongated by DNA polymerase III through the addition of nucleotides to the 3′-OH ends. On the
leading strand, DNA is synthesized continuously, whereas on the lagging strand, DNA is
synthesized in short stretches called Okazaki fragments. RNA primers within the lagging strand
are removed by the exonuclease activity of DNA polymerase I, and the Okazaki fragments are
joined by DNA ligase.
The histone chaperones involved in these processes are associated with replisome proteins:
CAF-1/Rtt106 with PCNA and FACT/Asf1 with MCMs.

2.) Transcription (DNA to RNA)

The general transcription factors comprise at least six distinct species: TFII A, B, D, E, F, and
H (see Fig. 7.1b). TFIID (300–750 kDa) is a multiprotein complex composed of a TATA (box)-
binding protein (TBP) and up to 13 TBP-associated factors (TAFs).
Transcription Factors and the Preinitiation Complex (PIC)

Unlike prokaryotic systems which can initiate the recruitment of RNAP

holoenzymes directly onto the DNA promoter regions and mediate the conversion
of RNAP to the open conformation, eukaryotic RNA polymerases require a host of
additional general transcription factors (GTFs), to enable this process. Here we will
focus on the activation of RNA Polymerase II as an example of the complexity of
eukaryotic transcription initiation.
Class II gene transcription in eukaryotes is a tightly regulated, essential process
controlled by a highly complex multicomponent machinery. A plethora of proteins,
more than a hundred in humans, are organized in often very large multiprotein
assemblies that include a core of General Transcription Factors (GTFs). The GTFs
include the factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNA polymerase
(RNA pol II), as well as a large number of diverse complexes that act as co-
activators, co-repressors, chromatin modifiers and remodelers (Fig. 10.10). Class II
gene transcription is regulated at various levels: while assembling on chromatin,
before and during transcription initiation, throughout elongation and mRNA
processing, and termination. A host of activators and repressors has been reported
to regulate transcription, including a central multi subunit complex called
the Mediator that helps in the recruitment of GTFs and the activation of RNA Pol
II. Here we will focus on the formation of the GTFs that make up the
core preinitiation complex (PIC) during transcriptional activation.

PIC contains, in addition to promoter DNA, the GTFs (TFIIA, B, D, E, F, and H),
and RNA Pol II. PIC assembly is thought to occur in a highly regulated, stepwise
fashion, as indicated. TFIID is among the first GTFs to bind the core promoter via
its TATA-box Binding Protein (TBP) subunit.

Transcriptional Elongation and Termination

Prokaryotic Transcriptional Elongation

The rate of transcription elongation by E. coli RNAP is not uniform. RNA synthesis
is characterized by pauses, some of which may be brief and resolved spontaneously,
whereas others may lead to the transcription elongation complex
(TEC) backtracking.

Elongation rate and pausing are determined by template sequence and RNA
structure (e.g., stem-loops) and involve at least two components of the RNAP
catalytic center, the bridge helix (BH) and trigger loop (TL). Elongation is
proposed to occur in three steps (Fig. 10.14). First, the TL folds in response to NTP
binding. Mutational analyses indicate that this conformational change in the TL can
be rate-limiting, and reflects the ability of the incoming NTP to bind to TEC. The
second step is the incorporation of the NTP and the release of pyrophosphate. The
third step involves the translocation of the RNAP down the DNA Template such
that the next RNA nucleotide can be added to the nascent transcript.

Figure 10.14 A model for Transcriptional Elongation. The trigger loop hinges,
bridge helix hinges and bridge helix bending models are based on all atom molecular
dynamics simulations. At the top of the figure, diagrams of the closed TEC, the
closed product TEC (after chemistry) and the translocating TEC are shown. DNA is
grey; RNA is red; the NTP substrate (or incorporated NMP and pyrophosphate) is
blue; the trigger loop (TL) is purple; the bridge helix (BH) is yellow. Interpretations
of simulations are shown schematically below. Simulations indicate trigger loop
hinges H1 and H2, bridge helix hinges H3 and H4 and bridge helix bend modes B1
(straighter) and B2 (more sharply bent).

Backtracking of TEC may take place after a brief pause in transcription, caused by
the thermodynamic properties of nucleic acids sequences surrounding the elongation
complex. In addition, misincorporation events render elongation complexes prone
to backtracking by at least one bp. In this case, the rescue from backtracking through
the cleavage of the 3′ end of the erroneous transcript also may be seen as a
proofreading reaction. Any backtracking event causes a pause or arrest of
transcription elongation, which may limit its overall rate (the average speed of
RNAP along the template) or processivity (the fraction of RNAP molecules reaching
the end of the gene).
While the general structure of the elongation complex (the transcription bubble, the
RNA-DNA hybrid) remains unchanged during backtracking, extension of RNA
becomes impossible in this conformation. However, such complexes can be resolved
by the hydrolytic activity of RNAP, which cleaves the phosphodiester bond in the
active center of the backtracked complex, producing a new RNA 3′ end in the active
center. For single base backups, the hydrolytic reaction is catalyzed by a flexible
domain of RNAP located in the secondary channel called the Trigger Loop (TL;
Figure 10.15B) and the two metal ions of the active center.
Longer sequences of backtracked TEC can restart when acted upon by GreA/B
factors, which restore the 3′-end of the nascent transcript to the active center. GreA
and GreB are transcript cleavage factors that act on backtracked elongation
complexes. When Gre factors are bound in the secondary channel, Gre factors
displace the TL from the active center (Figure 10.15). The displacement switches
off the relatively slow TL-dependent intrinsic transcript hydrolysis, and imposes the
highly efficient Gre-assisted hydrolysis. This efficiency is thought to be due to
stabilization of the second catalytic Mg2+ ion and an attacking water molecule by
the Gre factors.
Figure 10.15 The Role of Gre Factors in Relieving Transcription Elongation
Complex Backtracking. (A) Ribbon diagram of the GreA and GreB proteins. (B)
The mode of functioning of Gre factors. The Gre factor is bound to the active
elongation complex but does not impose hydrolytic activity on it. Upon backtracking
or misincorporation, the Gre factor protrudes its coiled-coil domain through the
secondary channel of RNAP (shown in the lefthand diagram), where it substitutes
for the catalytic domain Trigger Loop (TL). This substitution switches off the slow
TL-dependent phosphodiester bond hydrolysis and, and instead, facilitates highly
efficient Gre-dependent hydrolysis. After resolution of the backtracked complex
through RNA cleavage, the elongation complex returns to the active conformation
and the Gre factor gives way to the TL, which can now continue catalysis of RNA
synthesis (shown in the righthand diagram). The controlled switching between Gre
and the TL eliminates possible interference of Gre with the RNA synthesis.

Prokaryotic Transcriptional Termination

Transcription termination determines the ends of transcriptional units by
disassembling the transcription elongation complex (TEC), thereby releasing RNA
polymerases and nascent transcripts from DNA templates. Failure in termination
causes transcription readthrough, which yields wasteful and possibly harmful
intergenic transcripts. It can also perturb expression of downstream genes when the
unterminated TEC sweeps transcription initiation complexes off their promoters or
collides with RNA polymerases that transcribe opposite strands.
Transcriptional termination in prokaryotes can be template-encoded and factor-
independent (intrinsic termination), or require accessory factors, such as Rho, Mfd
and DksA. Intrinsic termination occurs at specific template sequences – an inverted
repeat followed by a run of A residues. Termination is driven by formation of a short
stem-loop structure in the nascent RNA chain (Figure 10.16). RNA synthesis arrests
and TEC dissociates at the 7th and 8th U of the run. Formation of the stem-loop
dissociates the weak rU:dA hybrid.
Stem-loop formation is hindered by upstream complementary RNA sequences that
compete with the downstream portion of the stem, as well as by RNA: protein
interactions in the RNA exit channel. Intrinsic termination depends critically upon
timing. Hairpin folding and transcription of the termination point must be
coordinated, so that the complete hairpin is formed by the time RNAP transcribes
the termination point. The size of the stem, the sequence of the stem and the length
of the loop all affect termination efficiency.

The bridge α-helix in the β’ subunit borders the active site and may have roles in
both catalysis and translocation. Mutations in the YFI motif (β’ 772-YFI-774) affect
intrinsic termination as well as pausing, fidelity and translocation of RNAP. One
mutation, F773V, abolishes the activity of the λ tR2 intrinsic terminator, although
neighboring mutations have little affect on termination. Modeling suggests that this
unique phenotype reflects the ability of F773 to interact with the fork domain in the
β subunit.

Figure 10.16 Model of Intrinsic Termination. (A) Shows the open conformation
of the RNAP during transcriptional elongation. RNAP is shown in yellow, DNA
template in blue, and nascent RNA in red. Key elements of the RNAP RNA exit
channel are shown in grey and labeled as indicated. (B) Shows the extension of the
nascent RNA through the RNAP exit channel and the potential for forming the RNA
hairpin structure when enough length has been achieved. (C) Shows the clamp
opening and disintegration of the the TEC when the RNA hairpin structure is
encountered at the transcriptional bubble.
Transcriptional termination can also be dependent upon accessory factors, such as
the Rho protein. Transcription termination factor Rho is an essential protein in E.
coli first identified for its role in transcription termination at Rho-dependent
terminators, and is estimated to terminate ~20% of E. coli transcripts. The rho gene
is highly conserved and nearly ubiquitous in bacteria. Rho is an RNA-dependent
ATPase with RNA:DNA helicase activity, and consists of a hexamer of six identical
monomers arranged in an open circle (Figure 10.17A).
Rho binds to single stranded RNA in a complex multi-step pathway that involves
two distinct sites on the hexamer. The primary binding site (PBS), distributed on the
N-terminal domains around the hexamer (Figure10.17B, cyan), ensures initial
anchoring of Rho to the transcript at a Rut (Rho utilization) site, a∼70 nucleotides
(nt) long, cytidine-rich and poorly-structured RNA sequence. Each Rho monomer
contains a subsite capable of binding specifically the base residues of a 5′-YC dimer
(Y being a pyrimidine). Biochemical and structural data suggest that Rho initially
binds to RNA in an open, ‘lock-washer’ conformation that closes into a planar ring
as RNA transfers to the central cavity. There, the ssRNA contacts an asymmetric
secondary binding site (SBS) (Fig. 10.17B, green), and this step, which presumably
is rate-limiting for the overall reaction, leads to motor activation. Upon hydrolysis
of ATP, the ssRNA is pulled upon conformational changes of the conserved Q and
R loops of the SBS, leading to Rho translocation, and ultimately promoting RNA
polymerase (RNAP) dissociation. The molecular mechanism of Rho translocation
based on single-molecule fluorescence methods appears to be tethered tracking. The
tethered tracking model postulates that Rho maintains its contacts between the PBS
and the loading (Rut) site upon translocation (Figure 10.17B). This mechanism
would allow Rho to maintain its high affinity interaction with Rut, and implies the
growing of an RNA loop between the PBS and the SBS upon translocation.

Figure 10.17 Schematic of Rho factor structure and mechanisms. (A) Molecular
structure of the Rho protein (PDB 1pv4) (B) Rho assembles as a homo-hexameric
ring (red spheres or tetragons), with RNA (black/yellow curve) binding to the
primary binding sites (PBS, cyan) and the secondary binding sites inside the ring
(SBS, green), where ATP-coupled translocation takes place. The Rut specific
binding site is depicted in yellow. The tethered-tracking model proposed that Rho
translocates RNA while maintaining interactions between PBS and Rut. This model
requires the formation of a loop that would shorten the extension of RNA upon
translocation.

Eukaryotic Transcriptional Termination

In eukaryotes, termination of protein-coding gene transcription by RNA polymerase
II (Pol II) usually requires a functional polyadenylation (pA) signal, typically a
variation of the AAUAAA hexamer. Nascent pre-mRNA is cleaved and the 5′
fragment is polyadenylated at the pA site shortly downstream from the hexamer by
cleavage and pA factors (CPFs). Two mechanisms have been suggested for pA-
dependent transcription termination. In the allosteric model, the pA signal and/or
other termination signals bind with the pA signal downstream region (PDR) and
induce reorganization of the Pol II complex. This includes the association or
dissociation of endonuclease components such as the CPFs. This causes
conformational changes in Pol II and TEC disassembly ensues. In the kinetic
model, also known as the “torpedo” model, cleavage at the pA site separates the
pre-mRNA from the TEC, which continues synthesizing a downstream nascent
transcript. This new transcript is a substrate of XRN2/Rat1p, a processive 5′-to-3′
exoribonuclease that catches up with, and disassembles, the TEC by an unknown
mechanism.
The two pA-dependent models are not mutually exclusive, and unified models have
been proposed. Loosely conserved pA signal sequences downstream of protein-
coding genes bind to components of the polyadenylation factor (CF1) complex
leading to assembly of the cleavage and polyadenylation machinery. Termination is
coupled to cleavage in a manner that has not yet been completely resolved, however,
one of the major factors involved in yeast pA termination is the endonuclease, Ysh1.
For example, the depletion of Ysh1 blocks TEC dissociation, but does not cause
substantial readthrough at the termination site (Fig. 10.18 A&B). These results
suggest that Ysh1 does not directly cause the pausing that occurs in the allosteric
termination pathway, but rather plays a role in the dissociation of the Pol II complex
from the DNA template (Figure 10.18A & B). It should be noted that not all pA-
dependent termination is dependent on Ysh1 and that other mechanisms of pA-
mediated termination still remain to be elucidated.
Figure 10.18 Schematic representation of Pol II termination after removal of
non-pA and pA termination factors. Elongating Pol II (green) terminates pA
transcripts (A) after an allosteric change (red) that reduces processivity. (B)
Depletion of Ysh1 leads to minimally extended readthrough transcripts but does not
block the allosteric change in Pol II. (C) Nrd1 and Nab3 binding recruit Sen1 for
termination of non-pA transcripts. (D) Pol II elongation complex lacking Nrd1 does
not recognize termination sequences in the nascent transcript and thus does not
facilitate the allosteric transition in Pol II. This leads to processive readthrough. (E)
Nrd1 and Nab3 recognize terminator sequences allowing the allosteric change in Pol
II but depletion of Sen1 blocks removal of Pol II from the template.

The mechanisms of termination of Pol II-mediated transcription differ for coding

and non-coding transcripts. Coding transcripts and possibly some stable
uncharacterized transcripts (SUTs) are nearly always processed at the 3′-end by the
cleavage and polyadenylation (pA) machinery and are processed by the pA-
dependent termination mechanisms described above. In contrast, ncRNAs are
terminated and processed by an alternative pathway that, in yeast, requires the RNA-
binding proteins Nrd1 and Nab3, as well as, the RNA helicase Sen1 (Fig 10.18 C).
Nrd1 and Nab3 recognize RNA sequence elements downstream of snoRNAs and
CUTs and this leads to the association of a complex that contains the DNA/RNA
helicase Sen1 and the nuclear exosome. The nuclear exosome is a complex of
ribonucleases with 3′ to 5′ exonuclease and endonuclease activity. It functions to
degrade unstable or incorrect RNA transcripts.
Both Nrd1 and Sen1 depletion lead to readthrough transcription of ncRNAs,
suggesting their importance in non-pA-dependent transcription termination (Fig
10.18 C & D). Furthermore, depletion of Nrd1 also causes the accumulation of
longer readthrough ncRNAs, suggesting its role in trafficking ncRNAs to the nuclear
exosome following termination.

10.5 Processing of RNA

Post-transcriptional modifications of rRNA and tRNA will be topics of Chapter 11
as their structure and function in protein synthesis will be a focal point. Thus, this
section will focus on post-transcriptional modifications of mRNA.
Prokaryotic RNA Processing
Bacterial cells do not have extensive post-transcriptional modification of mRNA
primarily because transcription and translation are coupled processes. Bacterial cells
lack the physical barrier of a nucleus, which allows transcription and translation
machineries to function at the same time, enabling the concurrent translation of an
mRNA while it is being transcribed (Fig 10.19). Within this system the NusG protein
plays a critical role. NusG has three separate domains and the functions of two of
them are known. The NusG N-terminal domain (NusG-NTD) has the capacity to
bind to RNAP, whereas the C-terminal domain (NusG-CTD) can combine with the
NusE (RpsJ) component of ribosomes. These two functions of NusG enable
transcription to be coupled with translation. NusG CTD can also bind to Rho to
terminate transcription (Figure 10.19).

Fig. 10.19. The roles of NusG in transcription/translation coupling. (a)

Composition of an active RNAP complex. RNAP is shown in dark grey, DNA in
blue and nascent RNA in red. The ribosome is shown in green with the nascent
polypeptide chain in light grey; the bulge in the small subunit denotes the location
of NusE (RpsJ). NusG is shown in orange: its shape denotes two functional sections.
The larger section denotes the N-terminal domain, which binds to RNAP. The
smaller section denotes the C-terminal domain, which interacts with NusE in situ.
Rho is shown in purple. (b) After translation is completed NusG remains bound to
RNAP and may also bind to Rho through the C-terminal domain leading to
termination of transcription.

Eukaryotic RNA Processing

In multicellular organisms almost every cell contains the same genome, yet complex
spatial and temporal diversity is observed in gene transcripts. This is achieved
through multiple levels of processing leading from gene to protein, of which RNA
processing is an essential stage. Following transcription of a gene by RNA
polymerases to produce a primary mRNA transcript, further processing is required
to produce a stable and functional mature RNA product. This involves various
processing steps including RNA cleavage at specific sites, intron removal,
called splicing, which substantially increase the transcript repertoire, and the
addition of a 5’CAP. Another crucial feature of the RNA processing of most genes
is the generation of 3′ ends through an initial endonucleolytic cleavage, followed in
most cases by the addition of a poly(A) tail, a process termed 3′ end cleavage and
polyadenylation (CPA).
3′-Polyadenylation
As seen in Section 10.4, polyadenylation is a required step for the correct
termination of nearly all mRNA transcripts. With the exception of replication
dependent histone genes, metazoan protein encoding mRNAs contain a uniform 3′
end consisting of a stretch of adenosines. In addition to deterimining the correct
transcript length at transcription termination, the poly(A) tail helps to ensure the
translocation of the nascent RNA molecule from the nucleus to the cytoplasm,
enhances translation efficiency, acts as a signal feature for RNA degradation, and
thereby contributes to the production efficiency of a protein.

CPA is carried out by a multi-subunit 3′ end processing complex, which involves

over 80 trans-acting proteins, comprised of four core protein subcomplexes (Figure
10.20 A). These consist of (1) cleavage and polyadenylation specificity factor
(CPSF), comprised of proteins CPSF1-4, factor interacting with PAPOLA and
CPSF1 (FIP1L1), and WD repeat domain 33 (WDR33) (shown in green on Figure
10.20 A); (2) cleavage stimulation factor (CstF), a trimer of CSTF1-3 (shown in red
on Figure 10.20 A; (3) cleavage factor I (CFI), a tetramer of two small nudix
hydrolase 21 (NUDT21) subunits, and two large subunits of CPSF7 and/or CPSF6
(shown in orange in Figure 10.20 A); and (4) cleavage factor II (CFII), composed of
cleavage factor polyribonucleotide kinase subunit 1 (CLP1) and PCF11 cleavage
and polyadenylation factor subunit (PCF11) (shown in yellow on Figure 10.20 A).
Additional factors include symplekin, the poly(A) polymerase (PAP), and the
nuclear poly(A) binding proteins such as poly(A) binding protein nuclear 1
(PABPN1).
CPA is initiated by this complex recognising specific cis-element sequences within
the nascent pre-mRNA transcripts termed polyadenylation signals (PAS). The PAS
sequence normally consists of either a canonical AATAAA hexamer, or a close
variant usually differing by a single nucleotide (e.g., ATTAAA, TATAAA). It is
located 10 to 35 nucleotides upstream of the cleavage site (CS) usually consisting a
CA dinucleotide. The PAS is also determined by surrounding auxiliary elements,
such as upstream U-rich elements (USE), or downstream U-rich and GU-rich
elements and G-rich sequences (DSE).
As soon as the nascent RNA molecule emerges from RNA polymerase II (RNA Pol
II), the CPSF complex is recruited to the PAS hexamer through numerous
interactions. Upon successful assembly of this macromolecular machinery, CPSF3
performs the endonucleolytic cleavage followed by a non-templated addition of
approximately 50-100 A residues.
Figure 10.20. The core 3′ end RNA processing machinery and impact on
alternative polyadenylation. (A) The core 3′ end processing machinery consists of
complexes composed of multiple trans acting proteins interacting with RNA via
multiple cis-elements (USE = upstream sequence element; PAS = poly(A) signal;
CS = cleavage site; DSE = downstream sequence element; CTD = C-terminal
domain). Upon co-transcriptional assembly of these complexes, RNA cleavage and
polyadenylation occurs to form the 3′ end of the nascent RNA molecule. (B) More
than 70% of all genes harbour more than one polyadenylation signal (PAS). This
gives rise to transcript isoforms differing at the mRNA 3′ end. While alternative
polyadenylation (APA) in 3′UTR changes the properties of the mRNA (stability,
localisation, translation), internal PAS usage (in introns or the coding sequence
(CDS)) changes the C-termini of the encoded protein, resulting in different
functional or regulatory properties.
Alternative polyadenylation (APA) occurs when more than one PAS is present
within a pre-mRNA and provides an additional level of complexity in CPA-
mediated RNA processing (Figure 10.20 B). Early studies revealed a significant
portion of genes undergo APA, and with the advent of next-generation RNA
sequencing technologies the large scale regulation of genes has become apparent,
with approximately 70% of the transcriptome exhibiting APA regulation. As APA
determines 3′UTR content and thus the regulatory features available to the mRNA,
changes in the APA profile of a gene can have enormous impacts on expression.
5′-CAP Formation
In eukaryotes, the 5′ cap, found on the 5′ end of an mRNA molecule, consists of a
guanine nucleotide connected to mRNA via an unusual 5′ to 5′ triphosphate linkage
(Fig. 10.21). This guanosine is methylated on the 7 position directly after capping in
vivo by a methyltransferase. It is referred to as a 7-methylguanylate cap, abbreviated
m7G.
In multicellular eukaryotes and some viruses, further modifications exist, including
the methylation of the 2′ hydroxy-groups of the first 2 ribose sugars of the 5′ end of
the mRNA. Cap-1 has a methylated 2′-hydroxy group on the first ribose sugar, while
cap-2 has methylated 2′-hydroxy groups on the first two ribose sugars. The 5′ cap is
chemically similar to the 3′ end of an RNA molecule (the 5′ carbon of the cap ribose
is bonded, and the 3′-OH unbonded). This provides significant resistance to 5′
exonucleases.
snRNAs contain unique 5′-caps. Sm-class snRNAs are found with 5′-
trimethylguanosine caps, while Lsm-class snRNAs are found with 5′-
monomethylphosphate caps. In bacteria, and potentially also in higher organisms,
some RNAs are capped with NAD+, NADH, or 3′-dephospho-coenzyme A. In all
organisms, mRNA molecules can be decapped in a process known as
messenger RNA decapping.
For capping with 7-methylguanylate, the capping enzyme complex (CEC) binds to
RNA polymerase II before transcription starts. As soon as the 5′ end of the new
transcript emerges from RNA polymerase II, the CEC carries out the capping
process (this kind of mechanism ensures capping, as with polyadenylation). The
enzymes for capping can only bind to RNA polymerase II that is engaging in mRNA
transcription, ensuring specificity of the m7G cap almost entirely to mRNA.
Figure 10.21 Structure of the 7-methylguanylate CAP.

The 5′ cap has four main functions:

1. Regulation of nuclear export
2. Prevention of degradation by exonucleases
3. Promotion of translation (see ribosome and translation)
4. Promotion of 5′ proximal intron excision
In addition to the polyA tail, nuclear export of RNA is regulated by the cap binding
complex (CBC), which binds to 7-methylguanylate-capped RNA (Fig 10.22). The
CBC is then recognized by the nuclear pore complex and the mRNA exported. Once
in the cytoplasm after the pioneer round of translation, the CBC is replaced by the
translation factors eIF4E and eIF4G of the eIF4F complex. This complex is then
recognized by other translation initiation machinery including the ribosome, aiding
in translation efficiency.
Capping with 7-methylguanylate prevents 5′ degradation in two ways. First,
degradation of the mRNA by 5′ exonucleases is prevented by functionally looking
like a 3′ end. Second, the CBC and eIF4E/eIF4G block the access of decapping
enzymes to the cap. This increases the half-life of the mRNA, essential in eukaryotes
as the export and translation processes take significant time.
The mechanism that promotes the 5′ proximal intron excision during splicing is not
well understood, but the 7-methylguanylate cap appears to loop around and interact
with the spliceosome, potentially playing a role in the splicing process.
Decapping of a 7-methylguanylate-capped mRNA is catalyzed by the decapping
complex made up of at least Dcp1 and Dcp2, which must compete with eIF4E to
bind the cap. Thus the 7-methylguanylate cap is a marker of an actively translating
mRNA and is used by cells to regulate mRNA half-lives in response to new stimuli.
During the decay process, mRNAs may be sent to P-bodies. P-bodies are granular
foci within the cytoplasm that contain high levels of exonuclease activity.

Figure 10.22 Importance of the 5-CAP during the lifespan of a mRNA

Transcript (a) CBC is required for pre-mRNA processing. The co-transcriptional
binding of CBC to 7mG prevents the decapping activities of pre-mRNA degradation
complexes [DXO (decapping exoribonuclease) and Dcp (decapping mRNA) Xrn2
(5′–3′ exoribonuclease 2)] and promotes pre-mRNA processing. CBC recruits P-
TEFb [Cdk9/Cyclin T1 (CycT1)] to transcription initiation sites of specific genes
promoting phosphorylation of the RNA pol II CTD at Ser2 residues. This results in
the recruitment of splicing factors including SRSF1, which regulates both
constitutive and alternative splicing events. Furthermore, CBC interacts with
splicing machinery components that results in the spliceosomal assembly. CBC
interacts with NELF and promotes pre-mRNA processing of replication-dependent
histone transcripts. (b) CBC forms a complex with Ars2 and promotes miRNA
biogenesis by mediating pri-miRNA processing. (c) CBC/Ars2 promotes pre-
mRNA processing of replication-dependent histone transcripts. (d) CBC promotes
export of U snRNA. CBC interacts with PHAX, which recruits export factors
including CRM1 and RAN·GTP. (e) CBC promotes export of mRNA. For export of
transcripts over 300 nucleotides, hnRNP C interacts with CBC and inhibits the
interaction between CBC and PHAX, allowing the CBC to interact with TREX and
the transcript to be translocated to the cytoplasm. CBC interacts with the PARN
deadenylase and inhibits its activity, protecting mRNAs from degradation. (f) CBC
mediates the pioneer round of translation. Cbp80 interacts with CTIF, which recruits
the 40S ribosomal subunit via eIF3 to the 5′ end of the mRNA for translation
initiation. Upon binding of importin-β (Imp-β) to importin-α (Imp-α), mRNA is
released from CBC and binds to eIF4E for the initiation of the standard mode of
translation. CBC-bound mRNP components not found in eIF4E-bound mRNPs are
CTIF, exon junction complex (EJC) and PABPN1. (g) The standard mode of
translation is mediated by eIF4E cap-binding protein. eIF4E is a component of the
eIF4F complex which promotes translation initiation.
Figure from: Gonatopoulos-Pournatzis, T. and Crowing V. (2014) Biochemical
Journal 457(2):231-42.
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mRNA Splicing
Eukaryotic genes that encode polypeptides are composed of coding sequences
called exons (ex-on signifies that they are expressed) and intervening sequences
called introns (int-ron denotes their intervening role). Transcribed RNA sequences
corresponding to introns do not encode regions of the functional polypeptide and are
removed from the pre-mRNA during processing. It is essential that all of the intron-
encoded RNA sequences are completely and precisely removed from a pre-mRNA
before protein synthesis so that the exon-encoded RNA sequences are properly
joined together to code for a functional polypeptide. If the process errs by even a
single nucleotide, the sequences of the rejoined exons would be shifted, and the
resulting polypeptide would be nonfunctional. The process of removing intron-
encoded RNA sequences and reconnecting those encoded by exons is called RNA
splicing. Intron-encoded RNA sequences are removed from the pre-RNA while it is
still in the nucleus. Although they are not translated, introns appear to have various
functions, including gene regulation and mRNA transport. On completion of these
modifications, the mature transcript, the mRNA that encodes a polypeptide, is
transported out of the nucleus, destined for the cytoplasm for translation. Introns can
be spliced out differently, resulting in various exons being included or excluded
from the final mRNA product. This process is known as alternative splicing. The
advantage of alternative splicing is that different types of mRNA transcripts can be
generated, all derived from the same DNA sequence. In recent years, it has been
shown that some archaea also have the ability to splice their pre-mRNA.
The splicing reaction is catalyzed by the spliceosome, a macromolecular complex
formed by five small nuclear ribonucleoproteins (snRNPs), termed U1, U2, U4, U5,
and U6, and approximately 200 proteins (Fig. 10.23). The assembly of the
spliceosome on pre-mRNA includes the binding of U1 snRNP, U2 snRNP, the pre-
formed U4/U6-U5 triple snRNP, and the Prp19 complex. This assembly occurs
through the recognition of several sequence elements on the pre-mRNA that define
the exon/intron boundaries, which include the 5′ and 3′ splice sites (SS), the
associated 3′ sequences for intron excision, the polypyrimidine (Py) tract, and the
branch point sequence (BPS). The assembly of the spliceosome during the process
is depicted in Figure 10.23.

Figure 10.23 Schematic representation of the spliceosome assembly and pre-

mRNA splicing. In the first step of the splicing process, the 5′ splice site (GU, 5′
SS) is bound by the U1 snRNP, and the splicing factors SF1/BBP and U2AF
cooperatively recognize the branch point sequence (BPS), the polypyrimidine (Py)
tract, and the 3′ splice site (AG, 3′ SS) to assemble complex E. The binding of the
U2 snRNP to the BPS results in the pre-spliceosomal complex A. Subsequent steps
lead to the binding of the U4/U5–U6 tri-snRNP and the formation of complex B.
Complex C is assembled after rearrangements that detach the U1 and U4 snRNPs to
generate complex B*. Complex C is responsible for the two transesterification
reactions at the SS. Additional rearrangements result in the excision of the intron,
which is removed as a lariat RNA, and ligation of the exons. The U2, U5, and U6
snRNPs are then released from the complex and recycled for subsequent rounds of
splicing.
Figure from: Suñé-Pou, M., et. al. (2017) Genes 8(3):87

In mammals, the first catalytic step of the splicing reaction begins when the U1
snRNP binds the 5′ SS of the intron (defined by the consensus sequence
AGGURAGU), and the splicing factors SF1 and U2AF cooperatively recognize the
BPS, Py, and 3′ SS to assembled complex E or the commitment complex (Figure
10.23). Subsequently, U2 snRNP and additional proteins are recruited to the pre-
mRNA BPS to form the pre-spliceosome or complex A. The binding of the U4/U6-
U5 tri-snRNP forms the pre-catalytic spliceosome or complex B. After RNA-RNA
and RNA-protein rearrangements at the heart of the spliceosome, U1 and U4 are
released to form the activated complex B or complex B* This complex is responsible
for executing the first catalytic step, through which the phosphodiester bond at the
5′ SS of the intron is modified by the 2′-hydroxyl of an adenosine of the BPS to form
a free 5′ exon and a branched intron (Fig. 10.24). The reaction of the 2′-hydroxyl
from the branchpoint adenosine nucleotide is known as a transesterification
reaction. During this process, additional rearrangements occur to generate the
catalytic spliceosome or complex C (Fig. 10.23), which is responsible for catalyzing
the second transesterification reaction leading to intron excision and exon–exon
ligation (Fig. 10.24). The resulting intron structure is referred to as a lariat structure.
After the second catalytic step, the U2, U5, and U6 snRNPs are released from the
post-spliceosomal complex and recycled for additional rounds of splicing.
Figure 10.24 Transesterification Reactions Involved in mRNA Splicing. (A)
Schematic diagram of the pre-mRNA with exons and introns indicated. Key
sequences are required for splicing at the 5′ and 3′ intron locations, and for the
recognition and positioning of the branchpoint Adenosine residue for the first
transesterification reaction. (B) Schematic of the two transesterification reactions
required for intron removal. The branchpoint 2′-OH residue mediates attack on the
5′-phosphate of the intron guanosine residue located at the 5′-splice site. This
releases the 3′ hydroxyl of Exon 1 which subsequently mediates attack of the 5′
phosphate of the first guanosine residue in Exon 2. The 3′ hydroxyl of the intron
guanine residue is released forming the Lariat structure and Exon 1 is ligated to Exon
2.

Alternative Splicing (AS) offers an additional mechanism for regulating protein

production and function. AS options are determined by the expression of or exposure
to in trans elements present within unique cellular locations and environments.
Additional sequence elements within the mRNA, known
as exonic and intronic splicing silencers or enhancers (ESS, ISS, ESE, and ISE,
respectively), participate in the regulation of AS. Specific RNA-binding proteins,
including heterogeneous nuclear ribonucleoproteins (hnRNPs) and serine/arginine-
rich (SR) proteins, recognize these sequences to positively or negatively regulate
AS (Figure 10.25). These regulators, together with an ever-increasing number of
additional auxiliary factors, provide the basis for the specificity of this pre-mRNA
processing event in different cellular locations within the body.

Figure 10.25 Alternative splicing (AS) regulation by cis mRNA elements

and trans-acting factors. The core cis sequence elements that define the
exon/intron boundaries (5′ and 3′ splice sites (SS), GU-AG, polypyrimidine (Py)
tract, and branch point sequence (BPS)) are poorly conserved. Additional enhancer
and silencer elements in exons and in introns (ESE: exonic splicing enhancers; ESI:
exonic splicing silencers; ISE: intronic splicing enhancers; ISI: intronic splicing
silencers) contribute to the specificity of AS regulation. Trans-acting splicing
factors, such as SR family proteins and heterogeneous nuclear ribonucleoprotein
particles (hnRNPs), bind to enhancers and silencers and interact with spliceosomal
components. In general, SR proteins bound to enhancers facilitate exon definition,
and hnRNPs inhibit this process. These trans-acting elements are expressed
differentially within different locations or under different environmental stimuli to
regulate AS.
Figure from: Suñé-Pou, M., et. al. (2017) Genes 8(3):87
There are several different types of AS events, which can be classified into four
main subgroups. The first type is exon skipping, which is the major AS event in
higher eukaryotes. In this type of event, a cassette exon is removed from the pre-
mRNA (Fig. 10.26 a). The second and third types are alternative 3′ and 5′ SS
selection (Fig. 10.26 b & c). These types of AS events occur when the spliceosome
recognizes two or more splice sites at one end of an exon. The fourth type is intron
retention (Fig. 10.26 d), in which an intron remains in the mature mRNA transcript.
This AS event is much more common in plants, fungi and protozoa than in
vertebrates. Other events that affect the transcript isoform outcome include mutually
exclusive exons (Fig. 10.26 e), alternative promoter usage (Fig. 10.26 f), and
alternative polyadenylation (Fig. 10.26 g).

Figure 10.26 Schematic representation of different types of alternative

transcriptional or splicing events, with exons (boxes) and introns
(lines). Constitutive exons are shown in green and alternatively spliced exons in
purple. Dashed lines indicate the AS event. Exon skipping (a); alternative 3′ (b) and
5′ SS selection (c); intron retention (d); mutually exclusive exons (e); alternative
promoter usage (f); and alternative polyadenylation (g) events are shown. Like
alternative splicing (AS), usage of alternative promoter and polyadenylation sites
allow a single gene to encode multiple mRNA transcripts.
(OTHERS)
Hypersensitivity key concepts:
Hypersensitivity reactions are exaggerated or inappropriate immunologic responses
occurring in response to an antigen or allergen.

Type I, II and III hypersensitivity reactions are known as immediate hypersensitivity

reactions because they occur within 24 hours of exposure to the antigen or allergen.