REVIEW

Alternative Polyadenylation: A Twist on mRNA

3= End Formation
Carol S. Lutz*
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey⫺New Jersey
Medical School, MSB E671, 185 South Orange Avenue, Newark, New Jersey 07101

R
NA is the intermediary in the information trans-
fer process, standing between the genetic mate- A B S T R A C T Regulation of gene expression by RNA processing mechanisms is
rial, DNA, and the end product, the translated now understood to be an important level of control in mammalian cells. Regula-
protein. Messenger RNA transcripts (mRNAs) in eukary- tion at the level of RNA transcription, splicing, polyadenylation, nucleo-cytoplasmic
otic cells have, with few exceptions, similar architecture transport, and translation into polypeptides has been well-studied. Alternative
and features. Most mRNAs have 5= and 3= untranslated RNA processing events, such as alternative splicing, also have been recognized
regions (UTRs), exons, and introns, usually thought of as as key contributors to the complexity of mammalian gene expression. Pre-
“coding” and “noncoding” regions, respectively, a spe- messenger RNAs (pre-mRNAs) may be polyadenylated in several different ways
cial modified base at the 5= end, called a “cap”, and a due to more than one polyadenylation signal, allowing a single gene to encode
number of signals present within the mRNA that signal multiple mRNA transcripts. However, alternative polyadenylation has only recently
translational “start” and “stop” (Figure 1). taken the field as a major player in gene regulation. This review summarizes what
Almost all eukaryotic mRNAs acquire an uncoded is currently known about alternative polyadenylation. It covers results from bioin-
poly(A) tail at their 3= ends in a process called polyadenyl- formatics, as well as those from investigations of viral and tissue-specific studies
ation (reviewed in refs 1⫺5). This process involves two and, importantly, will set the stage for what is yet to come.
tightly coupled steps, in which the mRNA is first cleaved
at a specific site, and then adenosine (A) residues are
added in a nontemplated fashion. The process of 3= end
formation is interconnected to other transcriptional and
post-transcriptional processes, such as splicing and
transcriptional termination (reviewed in refs 6⫺8). De-
fects in 3= end formation can drastically affect the devel-
opment, growth, and viability of a cell.
The Mechanism of Polyadenylation. Virtually all
mammalian polyadenylation signals contain the con-
sensus sequence AAUAAA (or a variant) between 10 and
35 nucleotides upstream of the actual cleavage and poly-
adenylation site (Figure 2; ref 9 and references therein).
In addition, sequences 14⫺70 nucleotides downstream *Corresponding author,
of the polyadenylation signal (the U/GU rich binding [email protected].
site for the multisubunit cleavage stimulation factor, or
CstF) are known to be involved in directing polyadenyl-
ation (10−22). These two elements are often termed the Received for review June 10, 2008
“core” polyadenylation elements. Auxiliary elements and accepted August 21, 2008.
both upstream and downstream of the core elements Published online September 26, 2008
have also been characterized that can enhance poly- 10.1021/cb800138w CCC: $40.75
adenylation efficiency and have been identified in both © 2008 American Chemical Society

www.acschemicalbiology.org VOL.3 NO.10 • ACS CHEMICAL BIOLOGY 609

 AAUAAA
AUG

STOP

Figure 1. A schematic of a mammalian pre-mRNA. Blue boxes indicate exons, and A single series of processing events may take place
black lines indicate introns. AUG indicates the translational start codon for the open on a pre-mRNA in the simplest of scenarios. However,
reading frame, and the stop sign indicates the translational stop codon. AAUAAA
indicates a polyadenylation signal, and the red baseball cap is representative of the
it is becoming increasingly clear that this simplest sce-
monomethylated guanosine cap added at the 5= end of the mRNA. Lighter blue re- nario is often not the rule. Alternative processing, that is,
gions represent the 5= and 3= UTRs. choice of one or more distinct processing events, of
mRNAs is now recognized as an important regulatory
viral and cellular systems (23−41). Spacing of these el- level in gene expression control (reviewed in refs 9 and
ements relative to the core elements is significant in 47⫺49). In alternative splicing events, different mRNA
their influence on polyadenylation (24, 26, 27, 30, 41). transcripts could be generated from alternative use of
Often these auxiliary elements are polyadenylation effi- splicing signals. Alternative polyadenylation is defined
ciency elements; however, they may also provide addi- as use of more than one polyadenylation signal. Taken
tional functions in proper processing. together, alternative processing events could result in
In addition to the RNA sequence elements, five multi- RNA transcripts that could differ in subtle ways, such as
subunit protein factors make up the core mammalian having different length 3= UTRs, or in more dramatic
polyadenylation and cleavage machinery: cleavage and ways, such as encoding different proteins with different
domains. These alternative processing events can then,
polyadenylation specificity factor (CPSF), CstF, cleavage
in turn, influence transcript localization, stability, and
factors Im and IIm (CF Im and CF IIm), and poly(A) poly-
transport. Although alternative splicing is an incredibly
merase (Figure 2; refs 1⫺5).The polyadenylation ma-
important mechanism of generating transcript diversity
chinery assembles on the pre-mRNA before any reac-
and in regulation of gene expression (reviewed in refs 47
tions take place. Cleavage occurs 10⫺30 residues after
and 48), this review will focus on the lesser-known and
the AAUAAA; the nuclease responsible for this specific
previously under-appreciated mechanism of alternative
cleavage has been reported to be CPSF 73 (42). polyadenylation and, in particular, alternative polyadenyl-
Polyadenylation and 3= end formation play a critical ation in mammals.
role in gene expression because improperly processed It has only been recently appreciated that more than
mRNAs are not transported out of the nucleus into the half of the genes in the human genome are alternatively
cytoplasm to be translated. Likewise, inefficiently pro- polyadenylated (9). Therefore, a closer understanding
cessed mRNAs may be transported out of the nucleus of alternative polyadenylation as a mechanism for tran-
with lower frequency than those mRNAs that are effi- script diversity and gene regulation is warranted. The
ciently processed. Poly(A) tails have also been shown current review is directed primarily at highlighting dis-
to influence mRNA stability, and translation (reviewed coveries on mammalian alternative polyadenylation that
in refs 43⫺46). Polyadenylation, therefore, is a funda- have been made in the past several years. The many
mental aspect of gene expression. consequences of alternative polyadenylation will be dis-
cussed at the end of this review, as
well as emerging questions in the
CFI, CFII, PAP,
field. Several other reviews (4, 5, 47)
symplekin, etc.
have discussed aspects of polyadenyl-
CPSF CstF ation and alternative polyadenyl-
ation, including the implications of
polyadenylation on health and dis-
ease (50). In addition, recent studies
AAUAAA U/GU rich on alternative polyadenylation in
Auxiliary upstream Core upstream Core downstream Auxiliary downstream plants and algae, specifically rice,
element element element element Arabidopsis, and Chlamydomonas, is
Cleavage site
yielding fascinating results that may
Figure 2. A schematic of RNA sequences and protein factors involved in polyadenylation. Green boxes
have important ramifications for our
are core polyadenylation elements; light green boxes are potential auxiliary elements that may or may understanding of mammalian alterna-
not be present in a polyadenylation signal. Ovals are basal polyadenylation machinery as indicated. tive polyadenylation (51−53).

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 REVIEW
Global Studies on Alternative Polyadenylation. With bound form (58). Now that alternative polyadenylation
the completion of the human genome sequencing is known to occur quite frequently, a number of studies
project, as well as other mammalian sequencing have shown dynamic interplay between these two pro-
projects, the opportunities for exploring and under- cesses (splicing and polyadenylation), the results of
standing polyadenylation and alternative polyadenyl- many of which lead to variant protein products (59, 60).
ation events expanded tremendously. Many of these It should also be noted that in one of the most com-
studies have used the large amount of available data mon techniques used to examine gene expression, the
such as expressed sequence tags (ESTs), cDNAs, or mi- microarray, resulting data can also be affected by alter-
croarray data in new and different ways, or developed native splicing and polyadenylation (61), depending on
new techniques such as tiling arrays to explore the com- the probe used for the microarray experiments. This
plex transcriptional landscape. One of the most striking could affect inaccurate interpretation of data from such
findings was that the number of transcripts encoded in experiments and underscores the need for further ap-
the mouse and human genomes were much larger, up preciation and study of alternative polyadenylation.
to 10 times or more, than the number of “genes” (refs Tissue-Specific Studies on Alternative Poly-
54 and 55 and references therein). This transcript varia- adenylation. Alternative polyadenylation of specific
tion represents, among other things, alternatively poly- mRNAs may be regulated differently in different tissues
adenylated mRNAs. in response to spatial, developmental, or functional
Several recent reports have examined mammalian needs. The question of tissue-specific alternative poly-
polyadenylation signals and patterns using available adenylation has recently generated a number of studies,
genomic information coupled with bioinformatics algo- some of which are bioinformatic reports, while other
rithms. These studies have determined that more than studies used more conventional molecular biology ap-
half of mammalian genes are alternatively polyadenyl- proaches. The bioinformatic approaches, by nature, ex-
ated and, furthermore, that many alternative polyadenyl- amined broad databases of information to search for
ation signals are evolutionarily conserved (9, 56, 57). patterns, as opposed to re-
The numbers are much higher than had previously been search on a specific gene or
expected, perhaps because less information was avail- family of genes. An early bioin- KEYWORDS
3= UTR: The noncoding or untranslated region at
able five or six years ago. What this means in the most formatic approach using avail- the 3= end of a mRNA. It immediately follows
simplistic of terms is that the mRNA landscape is richer able EST data (Oct 2000 the stop codon and includes regulatory
and more complex than previously anticipated. dbEST) and their program sequence elements, such as those that direct
formation of the poly(A) tail, as well as
The knowledge that alternative polyadenylation is termed ESTparser first sug- sequence elements that regulate mRNA
more widespread and complex than previously antici- gested a correlation between translation, mRNA stability, and binding sites
pated also impacts other studies of gene expression alternative polyadenylation for microRNAs (miRNAs).
mRNA: Messenger ribonucleic acid. mRNA is
regulation, for example, splicing. Alternative polyadenyl- and tissue- or disease-specific transcribed from DNA by an RNA polymerase.
ation may or may not be affected by splicing; type I poly- forms (62). Since then, EST da- Most mRNAs serve as templates for protein
adenylation is characterized by only one polyadenyl- tabases have improved and products. Mature mRNAs contain untranslated
regions at the 5= and 3= ends, as well as a
ation signal in an mRNA and is not affected by splicing grown such that a more com- modified base at the 5= end called a “cap”
(Figure 3; 9). Type II alternative polyadenylation as de- prehensive and refined picture and a poly(A) tail at the 3= end.
scribed by Tian et al. (9) has all alternative polyadenyla- is now being revealed. One re- Polyadenylation: The process by which an
unencoded polyadenylyl (poly(A)) chain is
tion signals in the 3= most exon and therefore is not af- cent study found that distinct added to the 3= end of a maturing mRNA. This
fected by splicing either (Figure 3). However, type III alternative polyadenylation reaction is mediated by multiprotein
alternative polyadenylation is characterized as having signal biases, that is, biased complexes, which first cleave the pre-mRNA
and subsequently add the poly(A) tail.
at least one alternative polyadenylation event coupled use of a particular alternative Alternative polyadenylation: Pre-mRNAs may be
with an alternative splicing event (Figure 3; 9). A classic polyadenylation signal, are polyadenylated in several different ways due
example of type III polyadenylation is the immunoglobu- found in reproductive tissues, to more than one polyadenylation signal,
allowing a single gene to encode multiple
lin mu system in B cells, where use of one polyadenyl- such as testis, uterus, and pla- mRNA transcripts. Sometimes these
ation signal during development leads to the secreted centa, and eye tissues, includ- transcripts differ only in their 3= end, and
form, while use of the other polyadenylation and the ac- ing the retina (63). Also, many sometimes they encode entirely different
proteins.
companying alternative splice leads to the membrane- interesting patterns of usage

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 AAUAAA
AUG

Type I STOP

only present in brain tissues in human, rat, and mouse

AAUAAA
AAUAAA
and an ⬃3⫺4 kb mRNA is found in other tissues (73).
AUG

STOP The alternative polyadenylation signal that is used in

Type II
this unusually long 3= UTR in the brain is highly con-
served over ⬃180 bases in these three species and
uses a suboptimal, conserved AGUAAA core upstream
element. In an interesting twist, a brain-specific pro-

AAUAAA
AAUAAA

moter was found in conjunction with the rat and mouse

AUG

STOP
Type III STOP ␤-adducin brain-specific alternatively polyadenylated
mRNAs, but this promoter is not found in humans (73).
The connection between cellular promoter choice and
polyadenylation signal choice has been further studied
Figure 3. Types of alternative polyadenylation. As described in ref 9, there are three in some other genes. Interestingly, a recent study sug-
basic types of alternative polyadenylation. Type I polyadenylation can be character- gests that tissue-specific expression of the X-linked
ized as constitutive polyadenylation, because only one polyadenylation signal is
gene MID1, involved in a malformation syndrome of
present in the 3= UTR, and is actually not alternative polyadenylation but is included
here for comparison. Type II alternative polyadenylation has more than one poly- midline structures called Opitz syndrome, is regulated
adenylation signal, but all polyadenylation signals are present in the 3= most exon. by concerted action of alternative promoters and alter-
Type III alternative polyadenylation involves alternative splicing coupled with native polyadenylation signals (74). In this study, it was
alternative polyadenylation. shown that cellular choice of a particular promoter dic-
were found in the brain (63) and in spermatogenesis tated which polyadenylation signal would define the 3=
(64). Finally, alternative polyadenylation patterns in end of the mRNA; however, a mechanism for how this
mammals seem to be conserved in evolution (56), sug- works is still unclear (74), although links between tran-
gesting selection pressure exists to keep these in place. scription initiation and transcription termination have
been discussed (7, 75).
These bioinformatic studies will provide an interesting
Finally, two recent studies report a connection be-
basis for validation in molecular biology laboratories in
tween tissue-specific alternative polyadenylation and
the near future.
translational efficiency (76, 77). A human apoptosis-
Tissue-specific polyadenylation of mRNA transcripts
inducing gene called hap was identified to have two dif-
has also been explored for a number of specific genes.
ferent sized mRNA transcripts, ⬃1.8 and ⬃2.7 kb, gen-
Although tissue-specific expression of some core poly-
erated by alternative polyadenylation (76). The larger of
adenylation factors themselves can and does occur
the two transcripts was found to be highest in brain and
(65−67), this has not yet been found to be a general
lowest in liver, while the ⬃1.8 kb transcript was high-
driving force in tissue-specific alternative polyadenyl-
est in testis and pancreas (76). Chloramphenicol acetyl-
ation. Here, I will mention a few examples of tissue- transferase (CAT) assays using reporter constructs con-
specific alternative polyadenylation of mRNA transcripts taining the polyadenylation signals from each of the two
from specific, noncore polyadenylation factor genes; transcripts showed that the ⬃2.7 kb transcript polyadenyl-
there are certainly many more that could be discussed. ation signal up-regulated translational activity ⬃3-fold,
Several examples are present in the recent literature while the overall levels of mRNA remained the same
demonstrating tissue-specific alternative splicing result- (76). Translational regulation coupled with alternative
ing in alternatively polyadenylated mRNAs (68−72). polyadenylation can have a negative effect as well.
These are all excellent examples of type III polyadenyl- BZW1 is a member of the bZIP superfamily of transcrip-
ation. Because of the recent nature of these studies, the tion factors, with a regular mRNA size of ⬃2.9 kb, but a
mechanism(s) of the alternative splicing (i.e., tissue- novel ⬃1.8 kb transcript was recently identified in the
specific expression of a regulatory splicing factor) have mouse testis (77). An AGUAAA suboptimal polyadenyl-
not yet been elucidated. ation signal is found 19 nucleotides downstream of the
A connection with transcription and alternative poly- stop codon, and use of this polyadenylation signal gives
adenylation has also been explored. The ␤-adducin rise to the testis-specific transcript (77). Most interest-
gene gives rise to two transcripts, an ⬃8⫺9 kb mRNA ingly, reporter constructs using this suboptimal poly-

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 REVIEW
adenylation signal, as compared with the optimal or interfere with polyadenylation as is the case for influ-
AAUAAA associated with the ⬃2.9 kb transcript, had enza virus (89−91).
the lowest translational efficiency. This is likely due to Alternative polyadenylation in HIV presents a differ-
different sequences in the 3= UTR, directed by the cho- ent set of interesting challenges because HIV viral RNA
sen polyadenylation signal, suggesting that choice of a sequences have repetitive regions at their 5= and 3=
particular polyadenylation signal may be able to regu- ends. Both ends contain polyadenylation signals, but
late translation of the mRNA. Although having a poly(A) the 3= polyadenylation signal would direct cleavage and
tail has long been correlated with efficient translation of polyadenylation of the mRNA transcript. Many studies
an mRNA, this particular connection has not been well over the years have postulated and demonstrated vari-
explored and warrants further study. ous reasons for why the 5= polyadenylation signal is not
Viral Studies on Alternative Polyadenylation. It has used, including promoter occlusion, lack of an up-
long been appreciated that viral genomes are often stream enhancer element, and presence of a major
very compact and use genetic information to its fullest splice donor site for U1snRNP binding, which is inhibi-
potential. Many viral genomes are highly alternatively tory (28, 34⫺36, and 92 and references therein). A new
spliced and polyadenylated. Recent studies describe twist was introduced in a recent study by the Shapiro
transcriptional profiles of previously uncharacterized vi- laboratory (93), which used computational folding pro-
ral transcripts. Transcripts from two parvoviruses, Aleu- grams to examine multiple strains of HIV-1 to make RNA
tian mink disease virus and Bocavirus bovine parvovi- structural predictions. They used the massively parallel
genetic algorithm (MPGA fold) to examine the folding
rus, have been recently characterized by the Pintel
propensities of the 5= and 3= poly(A) signals from nine
laboratory and show multiple, extensively processed
different HIV-1 strains and found that the 5= poly(A) sig-
mRNAs that are both alternatively spliced and poly-
nal is dominantly in a hairpin motif that inhibits prema-
adenylated, but in each case, they are derived from a
ture polyadenylation (93). This structural inhibition has
single transcriptional promoter (78, 79). Papillomavirus
previously been shown experimentally. The 3= poly-
type 16 (HPV16) and Kaposi’s sarcoma herpesvirus also
adenylation signal was shown, on the other hand, to
have many bicistronic or polycistronic transcripts that
have a propensity to be associated with a linear struc-
are widely processed through both alternative splicing
ture, which would also be more advantageous to poly-
and polyadenylation mechanisms (80−84). HPV16 has
adenylation signal use (93). These studies raise ques-
been well-characterized; it has two polyadenylation sig-
tions for future exploration of the role of structure in
nals, early and late, which are defined relative to their
polyadenylation signal choice, not only of viral mRNAs
temporal use during infection. The early polyadenyl- but also of cellular mRNAs.
ation signal is a type III polyadenylation signal and is Emerging Questions in the Field. While the observa-
found in an intron (reviewed in refs 81⫺84). The late poly- tions described here make it clearer than ever that alter-
adenylation signal of HPV16 is tightly linked to keratino- native polyadenylation is a powerful yet underexplored
cyte differentiation and is “off” at early times of infec- gene expression mechanism, there are many large ques-
tion and is turned “on” at late times. The on/off switch tions in the field that remain unanswered. Is the mech-
is controlled by a complex of cellular proteins that inter- anism of alternative polyadenylation different than con-
act with the HPV 3= UTR, including SF2/ASF, the stitutive polyadenylation, or does it use the same
U1snRNP, and CUG-BP1 (85−87). Avian retroviruses machinery? Are additional protein factors or sequence
have inefficient polyadenylation and are actually often elements involved, as in the case of alternative poly-
polyadenylated at the polyadenylation signal of down- adenylation of the COX-2 gene (26, 94) and prothrom-
stream host genes, yet another twist on the alternative bin (95, 96)? Many of the protein factors involved in al-
polyadenylation story (reviewed in ref 88). Although the ternative polyadenylation of COX-2 and prothrombin are
HPV16 story has been well-studied, it is not known or also splicing factors, suggesting further coordination of
yet fully appreciated in the cases of Kaposi’s sarcoma- polyadenylation and splicing events (94, 95). Other as-
associated and avian retroviruses the mechanisms by yet unexplored genes may also have a similar set of RNA
which alternative polyadenylation takes place in the vi- elements and RNA binding proteins that may regulate
rally infected cells, for example, if viral proteins promote their expression in a similar fashion. Do these factors in-

www.acschemicalbiology.org VOL.3 NO.10 • 609–617 • 2008 613

 teract with the basal polyadenylation machinery to ac- 3= UTRs, the connection is quite plausible and was re-
complish alternative polyadenylation? If so, how? If not, cently suggested to play a part in regulation of such glo-
is there an alternative machinery or parts of the machin- bal cellular programs as cellular proliferation (106).
ery? And how did alternative polyadenylation evolve in Ultimately, what are the consequences of alternative
the first place? Might alternative polyadenylation regu- polyadenylation? One consequence is the generation
late gene expression in some very interesting genetic of different mRNAs that encode different protein prod-
ways, such as the non-Mendelian inheritance mode ucts, such as would be the result of type III alternative
termed genomic imprinting, as recently suggested for polyadenylation. Examples include related proteins,
the mouse H13 gene (97)? Or might specific histone such as the immunoglobulin example given, but also
modification and chromatin architecture influence alter- very different proteins such as calcitonin and CGRP. An-
native polyadenylation (98)? And would these mecha- other straightforward consequence of alternative poly-
nisms be universal or gene-specific? adenylation is the prospect of generating mRNA prod-
It should also be considered that there may be an as- ucts with differential stability, which could ultimately
yet undeciphered “code” that may regulate aspects of
lead to more or less protein product, depending on en-
alternative polyadenylation. Candidates for such a code
hanced or diminished RNA half-life. Might alternative poly-
might include post-transcriptional modification of the
adenylation be a “rescue pathway” from nonsense-
core polyadenylation machinery, such as phosphoryl-
mediated decay surveillance, as suggested by Gilat and
ation or dephosphorylation (99, 100). A complex, com-
Shweiki (107)? It is appreciated that polyadenylation is
binatorial code involving RNA elements, RNA-binding
directly linked to transcription termination (reviewed in
proteins, and specific progesterone-mediated phosphor-
refs 8, 73, and 90), but how does alternative polyadenyl-
ylation/dephosphorylation of polyadenylation factors
ation interface with termination? Can alternative poly-
was recently described for the different yet related pro-
adenylation be post-transcriptionally modulated by
cess of cytoplasmic polyadenylation in Xenopus oocytes
(101). Might nuclear alternative polyadenylation use a nuclear mRNA retention and cell stress, as was sug-
similar complex but elegant code? gested for the CTN⫺mCAT2 gene pair (108)? Perhaps
More unanswered questions arise when we consider there are even more under-explored consequences of al-
the ever-expanding field of microRNA (miRNA) regula- ternative polyadenylation, including subcellular localiza-
tion of gene expression. miRNAs are small, noncoding tion and translational regulation. Taken together, the
RNAs that can regulate mRNA expression via up- or emerging topic of alternative polyadenylation has many
down-regulating transcription, splicing, translation, dif- interesting questions to address in the years to come.
ferentiation, and development (reviewed in refs Acknowledgment: The author wishes to thank Jeff Wilusz, Bin
102⫺105 and references therein). Might miRNAs also Tian, Lisa Canto, Sarah Darmon, and Catherine Newnham for
comments and critical reading of the manuscript. C.S.L. was sup-
be involved in regulating alternative polyadenylation? ported by National Science Foundation Award MCB-0462195
Because miRNA target binding sites are often found in and National Institutes of Health Award 5R03AR052038-03.

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