MBC 331

INTRODUCTION
All cells undergo a division cycle during their life span. Some cells are continually dividing (e.g. stem
cells), others divide a specific number of times until cell death (apoptosis) occurs, and still others
divide a few times before entering a terminally differentiated or quiescent state. Most cells of the
body fall into the latter category of cells. During the process of cell division everything within the cell
must be duplicated in order to ensure the survival of the two resulting daughter cells. Of particular
importance for cell survival is the accurate, efficient and rapid duplication of the cellular genome. This
process is termed DNA replication.

DNA Replication
Replication of DNA occurs during the process of normal cell division cycles. Because the genetic
complement of the resultant daughter cells must be the same as the parental cell, DNA replication
must possess a very high degree of fidelity. The entire process of DNA replication is complex and
involves multiple enzymatic activities.

The mechanics of DNA replication was originally characterized in the bacterium, E. coli which
contains 3 distinct enzymes capable of catalyzing the replication of DNA. These have been identified
as DNA polymerase (pol) I, II, and III. Pol I is the most abundant replicating activity in E. coli but has as
its primary role to ensure the fidelity of replication through the repair of damaged and mismatched
DNA. Replication of the E. coli genome is the job of pol III. This enzyme is much less abundant than
pol I, however, its activity is nearly 100 times that of pol I.

Up until a few years ago the use of Greek lettering to designate the six known eukaryotic DNA
polymerases was sufficient. However, recent evidence indicates that several more members of the
eukaryotic DNA polymerase family are present and function in distinct types of DNA replication.
These DNA polymerases are divided into four large families designated A, B, X, and Y. In addition,
there is a reverse transcriptase activity associated with telomerase and the mitochondrial polymerase
-primase enzyme. The original six distinct eukaryotic DNA polymerases are identified as α, β, γ, δ, ε,
and ζ. The identity of these individual enzymes relates to its subcellular localization, its primary
replicative activity and to the order in which it was first described.

The ability of DNA polymerases to replicate DNA requires a number of additional accessory proteins.
The combination of polymerases with several of the accessory proteins yields an activity identified as
DNA polymerase holoenzyme. These accessory proteins/complexes include (not ordered with
respect to importance):
1. Primase complex (DNA polymerase α complex)
2. Processivity accessory proteins
3. Single strand binding proteins, SSBP
4. Helicases
5. DNA ligases
6. Topoisomerases

The process of DNA replication begins at specific sites in the chromosomes termed origins of
replication, requires a primer bearing a free 3'–OH, proceeds specifically in the 5' → 3' direction on
both strands of DNA concurrently and results in the copying of the template strands in a semi-
conservative manner. The semi-conservative nature of DNA replication means that the newly
synthesized daughter strands remain associated with their respective parental template strands.
The large size of eukaryotic chromosomes and the limits of nucleotide incorporation during DNA
synthesis, make it necessary for multiple origins of replication to exist in order to complete
replication in a reasonable period of time. The precise nature of origins of replication in higher
eukaryotic organisms is unclear. However, it is clear that at a replication origin the strands of DNA
must dissociate and unwind in order to allow access to all of the accessory proteins and the DNA
polymerase complex. Unwinding of the duplex at the origin as well as along the strands as the
replication process proceeds is carried out by helicases. Helicases involved in DNA replication are
DNA-dependent ATPase with DNA helicase activity. The resultant regions of single-stranded DNA are
stabilized by the binding of single-strand binding proteins (SSBPs). The stabilized single-stranded
regions are then accessible to the enzymatic activities required for replication to proceed. The site of
the unwound template strands is termed the replication fork.

In order for DNA polymerases to synthesize DNA they must encounter a free 3'–OH which is the
substrate for attachment of the 5'–phosphate of the incoming nucleotide. During repair of damaged
DNA the 3'–OH can arise from the hydrolysis of the backbone of one of the two strands. During
replication the 3'–OH is supplied through the use of an RNA primer, synthesized by the activity of the
primase complex.

Synthesis of DNA proceeds in the 5' → 3' direction through the attachment of the 5'–phosphate of an
incoming dNTP to the existing 3'–OH in the elongating DNA strands with the concomitant release of
pyrophosphate. Initiation of synthesis, at origins of replication, occurs simultaneously on both
strands of DNA. Synthesis then proceeds bidirectionally, with one strand in each direction being
copied continuously and one strand in each direction being copied discontinuously. During the
process of DNA polymerases incorporating dNTPs into DNA in the 5' → 3' direction they are moving in
the 3' → 5' direction with respect to the template strand. In order for DNA synthesis to occur
simultaneously on both template strands as well as bidirectionally one strand appears to be
synthesized in the 3' → 5' direction. In actuality one strand of newly synthesized DNA is produced
discontinuously.

The strand of DNA synthesized continuously is termed the leading strand and the discontinuous
strand is termed the lagging strand. The lagging strand of DNA is composed of short stretches of
RNA primer plus newly synthesized DNA approximately 100–200 bases long (the approximate
distance between adjacent nucleosomes). The lagging strands of DNA are also called Okazaki
fragments. The concept of continuous strand synthesis is somewhat of a misnomer since DNA
polymerases do not remain associated with a template strand indefinitely. The ability of a particular
polymerase to remain associated with the template strand is termed its' processivity. The longer it
associates the higher the processivity of the enzyme. DNA polymerase processivity is enhanced by
additional protein activities of the replisome identified as processivity accessory proteins.

Diagrammatic representation of one side of a DNA replication fork. Large arrow depicts the overall
direction of replication with both the leading strand and lagging strands of replication shown
separated from each other. In actuality the process involves a looping of one of the two parental
strands in order to allow the simultaneous replication of both strands in what appears to be the same
direction. This detail is illustrated in the following Figure.

How is it that DNA polymerase can copy both strands of DNA in the 5' → 3' direction simultaneously?
A model has been proposed where DNA polymerases exist as dimers associated with the other
necessary proteins at the replication fork and identified as the replisome. The template for the
lagging strand is temporarily looped through the replisome such that the DNA polymerases are
moving along both strands in the 3' → 5' direction simultaneously for short distances, the distance of
an Okazaki fragment. As the replication forks progress along the template strands the newly
synthesized daughter strands and parental template strands reform a DNA double helix. The means
that only a small stretch of the template duplex is single-stranded at any given time.

Details of simultaneous DNA strand replication. Figure illustrates the mechanism by which both
strands of DNA are replicated simultaneously in the same direction. A portion of the lagging strand is
looped around through the DNA polymerase holoenzyme complex such that short stretches of
500–1000 can be continuously replicated in the same direction as the leading strand. Eventually
torsional stress will result in dissociation of the enzyme complex and the looping process will need to
begin again. This is what results in the average length of the Okazaki fragments generated from the
lagging strand. Only DNA helicase, single-strand binding proteins (SSBPs), and the DNA polymerase
complex are shown.

The progression of the replication fork requires that the DNA ahead of the fork be continuously
unwound. Due to the fact that eukaryotic chromosomal DNA is attached to a protein scaffold the
progressive movement of the replication fork introduces severe torsional stress into the duplex
ahead of the fork. This torsional stress is relieved by DNA topoisomerases. Topoisomerases relieve
torsional stresses in duplexes of DNA by introducing either double- (type II topoisomerases) or single-
stranded (type I topoisomerases) breaks into the backbone of the DNA. These breaks allow
unwinding of the duplex and removal of the replication-induced torsional strain. The nicks are then
resealed by the topoisomerases.

The RNA primers of the leading strands and Okazaki fragments are removed by the repair DNA
polymerases simultaneously replacing the ribonucleotides with deoxyribonucleotides. The gaps that
exist between the 3'–OH of one leading strand and the 5'–phosphate of another as well as between
one Okazaki fragment and another are repaired by DNA ligases thereby, completing the process of
replication.
Additional DNA Polymerase Activities

The main enzymatic activity of DNA polymerases is the 5' → 3' synthetic activity. However, DNA
polymerases possess two additional activities of importance for both replication and repair. These
additional activities include a 5' → 3' exonuclease function and a 3' → 5' exonuclease function. The
5' → 3' exonuclease activity allows the removal of ribonucleotides of the RNA primer, utilized to
initiate DNA synthesis, along with their simultaneous replacement with deoxyribonucleotides by the
5' → 3' polymerase activity. The 5' → 3' exonuclease activity is also utilized during the repair of
damaged DNA. The 3' → 5' exonuclease function is utilized during replication to allow DNA
polymerase to remove mismatched bases and is referred to as the proof-reading activity of DNA
polymerase. It is possible (but rare) for DNA polymerases to incorporate an incorrect base during
replication. These mismatched bases are recognized by the polymerase immediately due to the lack
of Watson-Crick base-pairing. The mismatched base is then removed by the 3' → 5' exonuclease
activity and the correct base inserted prior to progression of replication.

Post-Replicative Modification of DNA, Methylation

One of the major post-replicative reactions that modifies the DNA is methylation. DNA methylation
can alter chromatin structure, and can therefore, also alter the transcription of genes. The sites of
natural methylation (i.e. not chemically induced) of eukaryotic DNA is always on cytosine residues
that are present in CpG dinucleotides. However, it should be noted that not all CpG dinucleotides are
methylated at the C residue. The cytidine is methylated at the 5 position of the pyrimidine ring
generating 5-methylcytidine. Enzymes that incorporate methyl groups into DNA molecules are called
DNA methyltransferases, DNMTs.

Methylation of DNA in prokaryotic cells also occurs. The function of this methylation is to prevent
degradation of host DNA in the presence of enzymatic activities synthesized by bacteria called
restriction endonucleases. These enzymes recognize specific nucleotide sequences of DNA. The role
of this system in prokaryotic cells (called the restriction-modification system) is to degrade invading
viral DNAs. Since the viral DNAs are not modified by methylation they are degraded by the host
restriction enzymes. The methylated host genome is resistant to the action of these enzymes.

Following DNA replication the original pattern of methylation is copied by the maintenance methylase
enzyme, DNMT1. The DNMT1 protein recognizes the pattern of methylated C residues in the parental
DNA strand following replication and methylates the C residue present in the corresponding CpG
dinucleotide of the daughter strand.

Process of DNA methylation following DNA replication. Sites of DNA methylation have two fates following the
process of DNA replication: they can be maintained or they can be progressively removed. Following replication
the parental (template) strands of DNA contain 5mCpG, whereas the reciprocal C residue in the daughter strand
is not methylated. If the methylation state of the gene is to be maintained then the maintenance methylase,
DNMT1, incorporates a methyl group into the C residue of the daughter strand CpG dinucleotide.
DNA Recombination
DNA recombination refers to the phenomenon whereby two parental strands of DNA are spliced
together resulting in an exchange of portions of their respective strands. This process leads to new
molecules of DNA that contain a mix of genetic information from each parental strand. There are 2
main forms of genetic recombination. These are homologous recombination and non-homologous
recombination.

Homologous recombination is the process of genetic exchange that occurs between any two
molecules of DNA that share a region (or regions) of homologous DNA sequences. This form of
recombination occurs frequently while sister chromatids are paired during meiosis. Indeed, it is the
process of homologous recombination between the maternal and paternal chromosomes that
imparts genetic diversity to an organism. Homologous recombination generally involves exchange of
large regions of the chromosomes.

Holliday model of homologous recombination is the most common. In this model, recombination is
initiated by a pair of single-stranded breaks at homologous positions in two aligned DNA duplexes.
The strands of each duplex partly unwind, and each invades the opposite duplex, these strands can
be covalently joined to the opposite duplex creating a joint molecule in which two strands cross
between the DNA molecules. Branch migration then occurs: this is the simultaneous unwinding and
rewinding of the two duplexes such that the total number of hydrogen bonds remains constant, but
the position of the crossover moves. This creates a region of heteroduplex, a region where one strand
comes from the original duplex and the other strand comes from the other duplex. The two DNA
duplexes joined by a single crossover point can rotate to create a four-stranded holliday junction, it is
then resolved into two separate duplexes by either a vertical or horizontal cut.
Non-homologous recombination involves sequences that are not identical. Site-specific
recombination is an example of non-homologous recombination, and it involves involves exchange
between much smaller regions of DNA sequence (approximately 20–200 base pairs) and requires the
recognition of specific sequences by the proteins involved in the recombination process. Site-specific
recombination events occur primarily as a mechanism to alter the program of genes expressed at
specific stages of development. The most significant site-specific recombination events in humans
are the somatic cell gene rearrangements that take place in the immunoglobulin genes during B-cell
differentiation in response to antigen presentation. These gene rearrangements in the
immunoglobulin genes result in an extremely diverse potential for antibody production. A typical
antibody molecule is composed of both heavy and light chains. The genes for both these peptide
chains undergo somatic cell rearrangement yielding the potential for approximately 3,000 different
light chain combinations and approximately 5,000 heavy chain combinations. Then because any given
heavy chain can combine with any given light chain the potential diversity exceeds 10,000,000
possible different antibody molecules.

Integration of the bacteriophage λ into the Escherichia coli chromosome is the best studied example
of site-specific recombination. The λ-integrase catalyzes specific nicking of the bacteriophage DNA
and of a special sequence in the Escherichia coli chromosome, and it also catalyzes the resealing
involved.

Site-specific Recombination

Repair of Damaged DNA

Damage to DNA is a constant ongoing event and,
therefore, mechanisms are in place to constantly
surveil the structure of DNA and initiate the appropriate
response pathways to effect repair. Cancer, in most
non-viral induced cases, is the severe medically
relevant consequence of the inability to repair
damaged DNA. It is clear that multiple somatic cell
mutations in DNA can lead to the genesis of the
transformed phenotype. Therefore, it should be obvious
that complete understanding of DNA repair
mechanisms would be invaluable in the
design of potential therapeutic agents in the treatment
of cancer. DNA damage can occur as the result of
exposure to environmental stimuli such as alkylating
chemicals or ultraviolet and other forms of ionizing
radiation and free radicals (e.g. reactive oxygen
species, ROS) generated spontaneously in the
oxidizing environment of the cell. These phenomena
can, and do, lead to the introduction of mutations in the
coding capacity of the DNA. Mutations in DNA can
also, but rarely, arise from the spontaneous
tautomerization of the bases. The extent of
DNA damage that must be repaired is vast and
includes alterations to the DNA structure occurring during replication and recombination, damage to
the bases in the DNA (most often due to ROS and ionizing radiation), single- and double-strand breaks
in the backbone of the DNA, and strand cross-linking. Coding region mutations in DNA can be of two
general types. Transition mutations result from the exchange of one purine, or pyrimidine, for another
purine, or pyrimidine. Transversion mutations result from the exchange of a purine for a pyrimidine or
vice versa.

In order for the DNA damage repair systems to effect repair of DNA damage, the alterations need to
be recognized. Two major damage recognition systems function in human cells to effect DNA repair.
One is direct damage recognition and repair which refers to the mechanism by which the repair
enzyme or repair complex recognizes the damage and effects repair directly. An example of this type
of repair is described below in the context of the base excision repair (BER) process by which
alkylated guanine residues are recognized and repaired through the action of O6-methylguanine-DNA
methyltransferase. The second major mechanism is a multistep process where an enzyme or
complex recognizes the DNA damage and then recruits the repair enzyme or complex to the site of
damage to effect repair.

Base Excision Repair: BER

Base excision repair (BER) is a critical mechanism that ensures cells have a means to repair the vast
majority of endogenous DNA lesions. The types of DNA lesions that are corrected through the
process of BER include alkylations, oxidations, deaminations and depurinations, as well as single-
strand breaks (SSB). The primary function of BER is to remove these frequently produced lesions as a
means to ensure the integrity of the genome. The initial step in BER is the search for the lesions in
DNA by a family of DNA glycosylases which cut the N-glycosyl bond between the sugar and the base.
This step does not break the sugar-phosphate backbone of the DNA, rather it leaves an abasic
deoxyribose in the backbone that must be removed. Next, an AP (apurinic/apyrimidic) endonuclease
cleaves the phosphodiester bond at the 5’ side but leaves the sugar attached to the next nucleotide.
This is followed by the action of an AP lyase that cuts 3’ to the AP site to remove the sugar. The
resulting single nucleotide gap has a free 3’-hydroxyl. Finally, the gap is filled by DNA polymerase and
ligated by DNA ligase.

Nucleotide Excision Repair

Nucleotide excision repair (NER) involves the removal of a wide array of structurally unrelated DNA
lesions including cyclobutane thymine (pyrimidine) dimers (CPD), [6-4] pyrimidine-pyrimidine
photoproducts (6-4PP) produced in human skin by shortwave UV, and DNA helix-distorting chemical
adducts induced by xenobiotic chemical carcinogens. The prominent DNA adducts resulting from UV
irradiation of DNA are pyrimidine dimers, of which thymine dimers represent the most frequent
adducts formed. In addition to pyrimidine dimers the formation of 6-4PP adducts is also a common
result of UV irradiation. Pyrimidine dimers form from two adjacent pyrimidine residues in the same
strand of DNA.

The damage is removed by an enzyme complex that cuts several nucleotides away on both sides of
the damaged base(s), so the damage is released as part of an oligonucleotide. As in BER, the gap is
filled by DNA polymerase and ligated by DNA ligase.

DNA Mismatch Repair: MMR

DNA mismatch repair (MMR) is a highly conserved genome-maintenance system whose primary
function is to ensure replication fidelity by removing misincorporated bases and insertion-deletion
mispairs in newly synthesized DNA. Mismatch is a specialized form of nucleotide excision repair that
removes replication errors. Mismatches are not like DNA damage. There is no damage or modified
base present, just the wrong one of the four bases. Thus recognition of mismatches relies upon the
distortion of the double-helical structure. A major difference between the repair of DNA damage and
repair of mismatches is in the choice of base to excise, since both bases are normal. DNA in most
organisms is methylated at specific positions after replication, as post-replication modification.
During the very brief period where the daughter strand is not yet methylated, the mismatch repair
system can recognize the region of the mismatch and remove the nucleotide from the new strand.

In Escherichia coli, mismatches are recognized by a MutS homodimer, which creates a loop that
contains the mismatch. A MutL homodimer binds and coordinates the subsequent cleavage and
excision. Another protein, MutH nicks the unmethylated, newly synthesized strand on either sides of
the loop. The nicked single strand is degraded by an exonuclease. The long gap (hundreds of
nucleotides) that is created is filled by the action of DNA polymerase, and the remaining nick sealed
by DNA ligase.

Direct Demethylation

Modification of the DNA bases by alkylation (most often the incorporation of methyl groups) occurs
nearly exclusively on purine residues. Methylation of G residues allows them to base pair with T
instead of C. A unique activity called O6-methylguanine-DNA methyltransferase (encoded by the
MGMT gene) removes the methyl group from G residues. The protein itself becomes methylated and
is no longer active, thus, a single protein molecule can remove only one alkyl group. Another modified
guanine that is recognized as abnormal and removed is 8-oxoguanine (8-oxoG). The generation of 8-
oxoG occurs due to exposure of the DNA to reactive oxygen species, the consequences of which are
that 8-oxoG base pairs with A instead of C. The removal of 8-oxoG is catalyzed by the enzyme 8-
oxoguanine DNA glycosylase which is encoded by the OGG1 gene.

TRANSCRIPTION
Transcription is the mechanism by which a template strand of DNA is utilized by specific RNA
polymerases to generate one of the four distinct classifications of RNA. These four RNA classes are:

1. Messenger RNAs (mRNAs): This class of RNA is the genetic coding templates used by the
translational machinery to determine the order of amino acids incorporated into an elongating
polypeptide in the process of translation.

2. Transfer RNAs (tRNAs): This class of small RNA form covalent attachments to individual amino
acids and recognize the encoded sequences of the mRNAs to allow correct insertion of amino acids
into the elongating polypeptide chain.

3. Ribosomal RNAs (rRNAs): This class of RNA is assembled together with numerous ribosomal
proteins to form the ribosomes. Ribosomes engage the mRNAs and form a catalytic domain into
which the tRNAs enter with their attached amino acids. A unique function of the 28SrRNA of the large
ribosomal subunit is catalytic. This rRNA catalyzes the formation of the peptide bond via the
ribozyme (RNA-directed catalysis) activity.

4. Small RNAs: This class of RNA includes the small nuclear RNAs (snRNAs) involved in RNA splicing
and the microRNAs (miRNAs) involved in the modulation of gene expression through the alteration of
target mRNA activity.

All RNA polymerases are dependent upon a DNA template in order to synthesize RNA. The resultant
RNA is, therefore, complimentary to the template strand of the DNA duplex and identical to the non-
template strand. The non-template strand is called the coding strand because its sequences are
identical to those of the mRNA. However, in RNA, U is substituted for T and the intronic DNA
sequences are removed from the RNAs through the process of splicing.

Synthesis of RNA exhibits several features that are synonymous with DNA replication. RNA synthesis
requires accurate and efficient initiation, elongation proceeds in the 5' → 3' direction (i.e. the
polymerase moves along the template strand of DNA in the 3' → 5' direction), and RNA synthesis
requires distinct and accurate termination. Transcription exhibits several features that are distinct
from replication.

1. Transcription initiates, both in prokaryotes and eukaryotes, from many more sites than replication.

2. There are many more molecules of RNA polymerase per cell than DNA polymerase.

3. RNA polymerase proceeds at a rate much slower than DNA polymerase (approximately 50–100
bases/sec for RNA versus near 1000 bases/sec for DNA).

4. Finally the fidelity of RNA polymerization is much lower than DNA. This is allowable since the
aberrant RNA molecules can simply be turned over and new correct molecules made.

PROCESS OF TRANSCRIPTION

Signals are present within the DNA template that act in cis to stimulate the initiation of transcription.
These sequence elements are termed promoters. Promoter sequences promote the ability of RNA
polymerases to recognize the nucleotide at which initiation begins. Additional sequence elements are
present within genes that act in cis to enhance polymerase activity even further. These sequence
elements are termed enhancers. Transcriptional promoter and enhancer elements are important
sequences used in the control of gene expression.

The process of eukaryotic mRNA transcriptional initiation is an extremely complex event. There are
numerous protein factors controlling initiation, some of which are basal factors present in all cells
and others are specific to cell type and/or the differentiation state of the cell. Two basal promoter
elements that are found in essentially all eukaryotic mRNA genes are the TATA-box and the CAAT-
box.

Elongation involves the addition of the 5'–phosphate of ribonucleotides to the 3'–OH of the
elongating RNA with the concomitant release of pyrophosphate. Nucleotide addition continues until
specific termination signals are encountered. Following termination the core polymerase dissociates
from the template.
Transcription of mRNA

Transcriptional termination of eukaryotic mRNA genes occurs when RNA pol II encounters the
sequence, 3'-TTATTT-5', in the template DNA which directs the incorporation of the termination and
polyadenylation [poly(A)] signal, 5'-AAUAAA-3' in the mRNA. Following incorporation of the AAUAAA
element into the mRNA, the cleavage and polyadenylation specificity complex, which is associated
with the RNA polymerase complex, recruits other proteins to the site. The proteins that are recruited
then cleave the mRNA freeing it from the transcription complex and transcription terminates. RNA pol
II activity can be terminated by this process within 500–2,000 nucleotides of the AAUAAA element.

Co- and Post-transcriptional Processing of RNAs

Eukaryotic RNAs (all three classes) undergo significant processing, some of which occurs co-
transcriptionally and some post-transcriptionally. All three classes of RNA are transcribed from genes
that contain introns. The RNA sequences encoded by the intronic DNA must be removed from the
primary transcript prior to the RNA being biologically active. The process of intron removal is called
RNA splicing. Additional processing occurs to mRNAs that can alter the 5'- and 3'-ends of the
transcripts.

mRNA 5'-End Capping

The 5' end of nearly all eukaryotic mRNAs are capped with a unique 5' → 5' linkage to a 7-
methylguanosine residue. Synthesis of the mRNA cap structure is catalyzed by the bifunctional
enzyme encoded by the RNGTT gene (RNA guanylyltransferase and 5'-phosphatase). The capped end
of the mRNA is thus, protected from exonucleases and more importantly is recognized by specific
proteins of the translational machinery.
Structure of the 5'-cap of eukaryotic mRNAs. The cap structure present on most eukaryotic mRNAs
consists of a 7-methylguanosine (m7G) coupled to the 5'-terminal nucleotide of the mRNA in a unique
5' → 5' triphosphate linkage.

mRNA 3'-End Polyadenylation

Almost all mammalian mRNAs are polyadenylated at the 3'-end. A specific sequence, AAUAAA, is the
primary sequence recognized by one of several proteins and multiprotein complexes. In addition to
the AAUAAA sequence element in the mRNA, an upstream UGUA sequence and a downstream GU-
rich element act in cis to promote the recognition of the 3'-end of an mRNA by the cleavage and
polyadenylation complexes.

Humans express a family of three polyadenylate polymerases (PAP), These poly(A) polymerases
possess both mRNA endonuclease activity and polyadenylate polymerase activity. The endonuclease
activity cleaves the primary mRNA approximately 11–30 bases 3' of the AAUAAA sequence element.
A stretch of 20–250 adenosine residues is then added to the 3'-end by the non-template requiring
polyadenylate polymerase activity of the enzymes.

Both prokaryotic and eukaryotic rRNAs are synthesized as long precursors termed pre-ribosomal
RNAs. In eukaryotes a 45S pre-ribosomal RNA serves as the precursor for the 18S, 28S and 5.8S
rRNAs.

Splicing of RNAs

Spliceosome-Mediated RNA Splicing

The removal of intronic RNA from precursor mRNA, tRNA, and rRNA molecules, in humans and other
higher eukaryotes, requires a complex machinery termed the spliceosome which is composed of
numerous small nuclear RNAs (snRNAs) and numerous proteins. The spliceosome catalyzes the
reactions that result in intron removal and the joining together of the protein-coding exons. The
spliceosome has been shown to be composed of as many as 300 distinct proteins and five RNAs.
The five small nuclear RNAs (snRNAs) that constitute the spliceosome RNAs are identified as U1, U2,
U4, U5, and U6. Each of these snRNAs is around 100–300 nucleotides in length and each are
associated with several proteins forming individual small nuclear ribonucleoprotein (snRNP:
pronounced "snurp") complexes.

Introns in higher eukaryotic mRNAs can be of considerable length, in many cases spanning several
thousands of bases and sometimes comprising up to 90% of the precursor mRNA. In addition,
numerous precursor mRNAs undergo alternative exon splicing, a process controlled by many factors
such as the cell type in which the mRNA gene is expressed. Indeed, the vast majority of eukaryotic
mRNAs undergo some level of alternative splicing. The size and the number of introns in many
mRNAs, in addition to the potential for alternative splicing, present an array of complexities that
govern the control of, and catalytic processes of intron removal and exon joining.

Alternative Splicing

The process of alternative splicing involves multiple interactions between splicing proteins and
snRNPs that results in different patterns of exon joining from the same pre-mRNA in different cell
types or under different stages of development and differentiation. Alternative splicing allows for the
generation of protein isoforms that exhibit different biological properties, that differ in protein-protein
interaction, that are localized to different subcellular locations, or that exhibit different catalytic
activities and/or abilities. The process of alternative splicing has been identified to occur in the
primary transcripts from at least 80% of all human protein coding genes.

The molecular decisions that control which exon(s) is removed and which exon(s) is included in a
resultant mRNA involves both cis-acting RNA sequence elements and various protein regulators. The
overall process of alternative splicing requires that certain proteins are expressed that allow for
splice site recognition and selection as well as expression of proteins that inhibit splice site
recognition. In most cases of alternative splicing the regulation and specificity of which introns are
removed and which exons are joined together is the result of a combinatorial interaction between
both cis- and trans-acting activators and inhibitors.

Clinical Significances of Alternative and Aberrant Splicing

Abnormalities in the splicing process can lead to various disease states. Diseases that have been
identified as being due to alteration in, or the result of, alternative splicing are numerous. The causes
of the alterations in the alternative splicing process are also numerous. There are diseases that are
the result of mutations in splicing regulatory sequences in exons (e.g. the spinal muscular atrophies,
SMA) resulting in inappropriate exon skipping. Alterations in alternative splicing can also lead to
changes in protein isoform ratios that ultimately results in manifestation of disease (e.g. the diseases
of the brain that result from abnormal accumulation of the tau protein). Mutations in sequences
within introns can lead to the activation of cryptic splice sites resulting in abnormally spliced exons.
Numerous diseases are the result of mutations in either the 5'- or the 3'-splice sites such as various β-
thalassemias. Diseases are also caused by mutations in genes the encode proteins of the
spliceosomal machinery. Numerous human cancers are caused by mutations that alter splice site
selection, particularly in tumor suppressor genes, or by mutations in genes encoding protein factors
of the splicing machinery. Patients suffering from a number of different connective tissue diseases
exhibit humoral auto-antibodies that recognize small nuclear RNA-protein complexes (snRNPs).
Patients suffering from systemic lupus erythematosis (SLE) have auto-antibodies (anti-nuclear
antibodies) that recognize the U1 RNA of the spliceosome.