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Intron recognition comes of AGe

Melissa J. Moore

The molecular basis for the consensus sequence at the 3' ends of introns in higher eukaryotes has now been
elucidated. However, this discovery does not explain all aspects of 3' splice site selection.

Almost as soon as introns were discovered
in 1977, indelibly altering our view of
eukaryotic gene expression, the search was
on for factors that recognize and remove
them. In the ensuing years, our under-
standing of RNA splicing and its intrica-
cies have expanded enormously. Yet major
gaps have remained. One such gap has
© 2000 Nature America Inc. • http://structbio.nature.com
accurately on the nascent transcripts from
many different genes. This problem is
exacerbated in metazoans by the presence
of multiple introns per gene that often
account for much more of the initial tran-
script than the exons. It can be estimated
that the human spliceosome must be able
to recognize and remove somewhere
extending 10 or more nucleotides back into
the intron. The branch site is located
upstream of the PPT, generally 11–40
nucleotides from the 3' splice site YAG4,5.
Given the precise spacing between the PPT
and YAG, it seemed likely that both would
be key recognition elements for defining
the exact site of exon ligation during the

been a general lack of knowledge about
the factor(s) responsible for recognizing
and evolutionarily maintaining the con-
sensus sequence at the very 3' ends of
introns (Fig. 1a). Three papers in a recent
issue of Nature1–3 have taken a major step
toward filling this void.
The vast majority of introns are
removed by the major spliceosome, a 60 S
complex containing five small nuclear
RNAs (snRNAs: U1, U2, U4, U5 and U6)
and well over 50 different polypeptides
(Fig. 1b). Within this complex, intron
excision occurs in two steps, each a single
transesterification reaction. In the first
step, the 2'-OH of an adenosine in the
intron (the branch site A) attacks the
phosphodiester bond at the 5' splice site to
release the upstream exon and form a
branched or lariat splicing intermediate.
In the second step, the newly freed 3'-OH
of the upstream exon attacks the 3' splice
site phosphodiester to liberate the lariat
intron and join (ligate) the exons.
A major challenge for the splicing
machinery is to target these reactions
between 105 and 106 different intron second step of splicing. However, an assay
sequences. Thus, it is more than a little in which the sequence requirements specif-
disconcerting that the consensus ic to the second step could be monitored
sequences defining the sites of chemistry separately from those needed for the first
(Fig.1a) are not particularly information step revealed that accurate and efficient tar-
rich. Although mammalian introns can geting of the exon ligation reaction requires
exceed 100,000 nucleotides, reasonable only the YAG, and no attached PPT6. Thus,
matches to the consensus sequences at the something other than the active site for
5' splice site, branch site and 3' splice site exon ligation must be responsible for evo-
are predicted to occur every 290, 24, and lutionary maintenance of the PPT-YAG
490 nucleotides or so in random sequence, spacing, and this factor must act prior to
respectively4. To date the factors that the second step of splicing.
interact with and likely maintain evolu-
tionarily the 5' splice site and branch site
sequences have largely been defined.
Remarkably, however, even though the 3' b
splice site contains the most information,
the driving force behind this consensus
has been a mystery until now.
The mammalian 3' splice site consensus
can be broken down into two parts
(Fig. 1a): a highly conserved YAG (where Y
is a pyrimidine) at positions -1 to -3 relative
to the site of exon ligation, and a stretch of
pyrimidines (known as the polypyrimidine
tract or PPT) beginning at position -5 and

Fig. 1 a, Consensus sequences found in human introns. Y, R, and N, indicate pyrimidine, purine
and any nucleotide, respectively. The most highly conserved positions are shown in red and the
polypyrimidine tract in blue. b, The two steps of splicing mediated by the spliceosome (lavender).
In both parts, the intron is represented by a curved line and exons by green boxes.

14 nature structural biology • volume 7 number 1 • january 2000

 © 2000 Nature America Inc. • http://structbio.nature.com
Curiously, even though the spacing
between the PPT and branch site is more
variable than that between the PPT and
YAG, the PPT had been singled out early
on as an important recognition element
for branch site definition prior to the first
step of splicing. The factor responsible for
this early PPT recognition is U2AF65, a
subunit of the U2 snRNP auxiliary factor
(U2AF). U2AF was originally defined as a
biochemical activity required for the addi-
tion of U2 snRNP to the branch site at an
early stage of spliceosome assembly.
Proper spliceosome assembly and subse-
quent splicing depends on base pairing
between a sequence in U2 snRNA and the
branch site consensus. Native U2AF is a
heterodimer of 65 and 35 kDa subunits.
Because recombinant U2AF65 (rU2AF65)
© 2000 Nature America Inc. • http://structbio.nature.com
alone could rescue in vitro splicing of
some introns in extracts depleted for the
endogenous U2AF heterodimer, this sub-
unit has been the focus of intensive
research over the years to define its struc-
ture, its role in branch site definition, and
its RNA binding properties (for refer-
ences, see 1–3, 7–9). U2AF65 contains
three prototypical RNA recognition motif
(RRM) domains for binding RNA, as well
as a region rich in arginines and serines
(the RS domain) that functions in U2
snRNP recruitment. However, the RNA
binding properties of U2AF65 could not
entirely explain the long mammalian 3'
splice site consensus: SELEX (systematic
evolution of ligands by exponential
enrichment) revealed that U2AF65 has
high affinity for pyrimidine-rich
sequences alone, and not for a PPT fol-
lowed by a YAG10 (Fig. 2a, left). Thus, a
key piece in the 3' splice site sequence puz-
zle was clearly missing.
The groups of Blumenthal, Green,
Nilsen and Varcárcel have now identified
this missing piece. These papers show that
the smaller U2AF subunit, U2AF35, which
contains no known RNA binding motif,
can be crosslinked to the 3' splice site AG
in extracts from organisms as diverse as
worms, fruit flies and humans1–3. This
interaction is specific because disruption
of the AG dramatically reduced crosslink
yields in all cases. Remarkably, a SELEX
experiment performed with the human
U2AF heterodimer (that is, U2AF65 and
U2AF35 together) yielded a sequence that
is almost exactly the mammalian 3' splice
site consensus1 (Fig. 2a, right). This select-
ed consensus sequence not only repro-
duces the distinct spacing between the
PPT and YAG, but it also recapitulates the
preference for G and U as the first two
nucleotides of the downstream exon
nature structural biology • volume 7 number 1 • january 2000
news and views
(Fig. 1a). Therefore, recognition by the cific 3' splice sites by the Sex-lethal (SXL)
U2AF heterodimer early in spliceosome protein present only in female embryos.
assembly is most certainly the evolution- SXL is an RRM-containing protein with a
ary driving force behind the extended similar but distinct affinity for pyrimidine-
mammalian 3' splice site consensus rich regions as U2AF65 (ref. 10), but it lacks
sequence. the RS domain in U2AF65 required to
Another mystery solved by the recruit U2 snRNP to the branch site. Thus,
U2AF35–3' splice site AG interaction con- SXL can block splicing of an intron simply
cerns ‘AG-dependent’ versus ‘AG-indepen- by occupying the PPT and preventing the
dent’ introns11. It was previously known binding of U2AF. The Valcárcel group3 has
that mammalian introns with short or now elucidated how the structure of a reg-
weak PPTs require the 3' splice site AG for ulated 3' splice site in the msl-2 gene has
both spliceosome assembly and lariat for- been optimized to enable SXL to compete
mation (Fig. 2b, right), whereas these with U2AF. Because the 3' splice site AG is
processes can occur in the absence of a 3' not immediately adjacent to the PPT in
splice site AG on introns with long PPTs this intron, U2AF is limited to interactions
(Fig. 2b, left). Several groups have now with the PPT alone; this weakens U2AF
demonstrated that in extracts depleted of binding and allows SXL to compete effi-
the endogenous U2AF heterodimer, splic- ciently (Fig 3a). However, when the AG
ing of AG-dependent introns can be res- was moved closer to the PPT to strengthen
cued only when both rU2AF65 and the binding of U2AF, SXL could no longer
rU2AF35 are added, and not with rU2AF65 compete and splicing proceeded even in
alone1,7,12. This is consistent with gel shift the presence of normally inhibitory SXL
and binding competition assays indicating concentrations (Fig. 3b).
that the presence of an AG adjacent to the Although interaction of the U2AF het-
PPT significantly strengthens RNA bind- erodimer with the full 3' splice site con-
ing by the U2AF heterodimer1–3. sensus sequence including the YAG is
The paper by Meredino et al.3 illustrates clearly crucial for recruitment of U2
how cells can capitalize on the relatively snRNP to the branch site on some introns,
weaker binding of U2AF to a PPT with no this interaction is not necessarily what
adjacent AG to mediate regulated splicing. specifies the exact phosphodiester bond
Sex determination in Drosophila involves a used as the site of exon ligation. Several
cascade of alternative splicing events. results are consistent with this idea. First,
Several of these involve inhibition of spe- the AG that functions to promote spliceo-
a
b
Fig. 2 a, Consensus binding sites for the U2AF65 monomer (left) and U2AF65/35 heterodimer (right)
determined by iterative in vitro selection. b, In AG-independent introns (left), U2AF65 can bind the
long PPT (blue box) tightly enough on its own to recruit U2 snRNP to the branch site. However,
when the PPT is short (right), additional binding interactions between U2AF35 and an adjacent AG
are required for stable U2AF association and U2 snRNP recruitment. Thus the latter introns are
said to be ‘AG-dependent’.
15
 © 2000 Nature America Inc. • http://structbio.nature.com

news and views

a such complexes. So early recognition of

the 3' splice site AG is a likely means by
which cells control the fidelity of intron
recognition.
In summary, the new findings1–3 regard-
ing interaction of U2AF35 with the 3' splice
site AG have finally explained the evolu-
tionary driving force behind the mam-
malian 3' splice site consensus. Thus, the
3' splice site AG is an important recogni-
tion element for defining both the branch
b site prior to the first chemical step of splic-
ing and the site of exon ligation prior to
the second chemical step. However, the
U2AF35–AG interaction early in spliceo-
some assembly is just the beginning of a
complex process by which the eventual
site of exon ligation is determined. The
molecular details of this process await fur-
© 2000 Nature America Inc. • http://structbio.nature.com

ther experimentation.

Melissa J. Moore is in the Department of

Fig. 3 A longer than usual distance between the PPT and 3' splice site AG in the wild type msl-2
Biochemistry, Howard Hughes Medical
intron allows for inhibition of splicing by the Drosophila Sex-lethal (SXL) protein. a, Because the Institute, Brandeis University, 415 South
AG in the wild type msl-2 intron is out of reach of U2AF35, binding of the U2AF heterodimer to the Street, Waltham, Massachusetts, 02254-
PPT (blue box) is less stable. Thus, the SXL protein present in female embryo cells can successfully 9110, USA. email: mmoore@brandeis.edu
compete for PPT binding and thereby block msl-2 splicing. b, If, however, the 3' splice site AG is
moved adjacent to the PPT, the U2AF heterodimer binds so tightly that SXL cannot compete, so
splicing occurs. The predominant protein–RNA complex in each panel is indicated by a shaded box.
1. Wu, S., Romfo, C.M., Nilsen, T.W. & Green, M.R.
Nature 402, 832–835 (1999).
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(1999).
3. Merendino, L., Guth, S., Bilbao, D., Martinez, C. &
Valcárcel, J. Nature 402, 838–841 (1999).
some assembly can be different from the non-Watson-Crick interaction between 4. Burge, C.B., Tuschl, T. & Sharp, P.A. In The RNA
one used for exon ligation13. Second, the Gs at the 5' and 3' termini of introns world, second edition. (eds. Gestland, R.F., Cech,
T.R. & Atkins, J.F.) 525–560 (Cold Spring Harbor
U2AF reportedly dissociates from mam- (refs 4,8,15,17–19 and refs therein). Most Press, Cold Spring Harbor, New York; 1999).
malian splicing complexes after U2 recently, Chua and Reed20 reported that 5. Senapathy, P., Shapiro, M.B. & Harris, N.L. Methods
Enzymol. 183, 252–278 (1990).
snRNP addition but before the first chem- in vitro depletion of the human Slu7 pro- 6. Anderson, K. & Moore, M.J. Science 276, 1712–1716
ical step of splicing14. This is well before tein results in promiscuous 3' splice site (1997).
7. Guth, S., Martinez, C., Gaur, R.K. & Valcárcel, J. Mol
the exact site of exon ligation is deter- AG choice at the time of exon ligation. Cell Biol 19, 8263–8271 (1999).
mined, which need not occur until after Moreover, some sort of linear search 8. Krämer, A. Annu. Rev. Biochem. 65, 367–409 (1996).
9. Cáceres, J.F. & Krainer, A.R. In Eukaryotic mRNA
the first step15. Third, there is apparently mechanism has been implicated in iden- processing. (ed. Krainer, A.R.) 174–212 (Oxford
no early recognition of the 3' splice site AG tifying the exact site of exon ligation University Press, Oxford; 1997).
10. Singh, R., Valcárcel, J. & Green, M.R. Science 268,
in budding yeast; neither mutation of the when no AG is close to the branch site 1173–1176 (1995).
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the branch site inhibits in vitro lariat for- say that U2AF has no part in determining (1996).
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14. Bennett, M., Michaud, S., Kingston, J. & Reed, R.
tent with there being no protein with early interaction with the 3' splice site AG Genes Dev. 6, 1986–2000 (1992).
obvious sequence similarity to U2AF35 in would be an efficient means to ensure 15. Anderson, K. & Moore, M.J. RNA 6, 1–10 (2000).
16. Rymond, B.C. & Rosbash, M. Nature 317, 735–737
Saccharomyces cerevisiae . that any branch site chosen will have a (1985).
Unlike U2AF35, numerous spliceosome potential exon ligation site nearby. At 17. Zhou, Z. & Reed, R. EMBO J. 17, 2095–2106 (1998).
18. Schwer, B. & Gross, C.H. EMBO J 17, 2086–2094
components that are conserved between least in vitro, spliceosomes that have car- (1998).
yeast and humans have been implicated ried out the first step of splicing on an 19. Collins, C.A. & Guthrie, C. Genes Dev. 13, 1970–1982
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in choosing the 3' splice site AG prior to AG-independent intron lacking a proper 20. Chua, K. & Reed, R. Nature 402, 207–210 (1999).
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Prp22, Slu7, U5 and U6 snRNAs, and a interest of the living cell to accumulate 3356–3367 (1994).

16 nature structural biology • volume 7 number 1 • january 2000

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