Introns with splice-site sequences with poor matches to the consensus motifs are common in pre-mRNAs. Despite having weak splicing signals, such introns can be excised efficiently in vivo. The mechanisms responsible for the recognition and selection of authentic splice sites, rather than cryptic sites or alternative splicing pathways, are not clear. In particular, the highly specific recognition of 3′ splice sites is puzzling. The consensus sequence of the 3′ splice site is relatively simple, apparently requiring little more than an AG dinucleotide preceded by a region moderately enriched in pyrimidines and a degenerate branchpoint sequence. Clearly, much more is involved in defining a 3′ splice site, as this combination of sequence elements is plentiful in genomic sequences and yet 3′ splice-site selection is highly specific and subject to inactivation by single point mutations. It is apparent that our current understanding of the 3′ splice site is limited, and has likely overlooked components that may be critical for the efficient and specific recognition of sites that have poor matches to the degenerate 3′ splice-site consensus.
To gain insight into the mechanism of 3′ splice-site recognition, we investigated the factor requirements for splicing of an intron with a weak 3′ splice site/pyrimidine tract, and identified PUF60 as a critical protein for splicing (). We showed that PUF60 stimulates splicing, and is in a complex with splicing factors involved in the early steps of spliceosome assembly (). Mechanistically, PUF60 collaborated in a cooperative manner with U2AF65/35 in both RNA binding ( and ) and splicing activation (). Surprisingly, however, neither protein was essential for splicing, provided that the other one was present indicating some level of functional redundancy between these structurally related proteins. In addition, modulation of the levels of U2AF65 and PUF60 in cells changed alternative splicing patterns ( and ), demonstrating that PUF60 and U2AF65 can modulate the efficiency of 3′ splice-site selection in a splice-site-dependent manner, and thereby regulate alternative splicing.
PUF60: a Multi-tasking Protein
PUF60 has long been considered a putative splicing factor due to its presence in a number of purified spliceosomes (reviewed in 
), its similarity to U2AF65 
, as well as its presence in a partially purified fraction of nuclear extract with splicing activity in vitro 
. In the latter study, PUF60 was shown to bind to poly(U) RNA and was the predominant protein along with SRp54 in a partially purified fraction of nuclear extract that complemented splicing of extract depleted of poly(U) binding factors. However, none of the functional assays done at that time used recombinant PUF60. Thus, despite the suggestive evidence that PUF60 was a splicing factor, rigorous proof of its function in splicing was previously lacking. To demonstrate the activity of PUF60 in splicing, we have used an S100 extract complementation assay, as well as the previous assay involving complementation of poly(U)-depleted extracts. For the latter assay, we used different substrates than Page-McCaw et al. 
, as well as recombinant PUF60 protein purified from mammalian 293 cells for our complementation; thus, it is possible that our PUF60 protein is more active and/or our splicing substrates may be more efficient or responsive to PUF60 activity.
PUF60 has other documented roles in the cell, and appears to be a protein with particularly diverse functions. PUF60 is also known as FBP-interacting repressor (FIR), a regulator of Myc gene expression 
. In this role, PUF60/FIR represses Myc transcription in a process that involves binding between FIR and FUSE-binding protein (FBP), which binds the Myc promoter region. FIR/PUF60 itself was not found to bind the DNA, but instead enhanced FBP binding. We did not detect an association between FBP and PUF60 by western (data not shown) or by mass-spectrometry of PUF60 immunoprecipitations. However, this result does not preclude a relationship between these two proteins under other conditions. A role for PUF60 in transcription as well as splicing is intriguing, as this implies that PUF60 could contribute to the coupling between these two processes (reviewed in 
Finally, PUF60 is also known as RoBPI (Ro RNA binding protein) and interacts with Ro ribonucleoproteins (RNPs) 
. Ro RNPs have largely unknown functions, but are currently thought to play a role in quality control of small RNAs (reviewed in 
Cooperation between PUF60 and U2AF65/35 has a General Role in Splicing
The PyD splicing substrate with a weak pyrimidine tract was used to initially identify PUF60 as a splicing factor. Previous analysis of this substrate revealed that it requires both U2AF65
for splicing, whereas splicing of the wild-type parental substrate is not dependent on U2AF35
, Hastings&Krainer, unpublished results). U2AF35
recognizes the 3′ splice-site AG dinucleotide and stabilizes binding of U2AF65
to the pyrimidine tract 
. This role for U2AF35
may be particularly important in substrates with weak pyrimidine tracts that are not bound efficiently by U2AF65
. Similarly, PUF60 may be required in addition to U2AF65
to facilitate splice-site recognition. In these instances, in which splicing is inefficient, the synergistic activity of these proteins may be critical for splice-site identification.
We propose that 3′ splice-site selection efficiency is dictated in part by the ability of the site to be recognized by U2AF65/35
and PUF60. Splicing efficiency, as well as alternative splicing patterns, could thereby be dictated by the availability, modifications, or expression levels of these proteins. One possible function of the proteins may be to displace inhibitory factors from the pyrimidine tract. Indeed it has been reported that modulation of the levels of U2AF65
and the inhibitory protein PTB (polypyrimidine-tract-binding protein) can influence alternative 3′ splice-site selection 
Mechanistic Considerations for PUF60 in Splicing
Cooperation between PUF60 and U2AF65/35
was observed for all the splicing substrates tested, suggesting that this activity is an integral part of the splicing process. Synergy between proteins in splicing may reflect cooperative binding to a functional element(s), or multiple, simultaneous interactions between the activators and other components of the splicing machinery (reviewed in 
). Indeed, we find that having both PUF60 and U2AF65/35
present not only stimulates splicing in vitro
in a cooperative manner, but also influences their binding to the 3′ splice-site region ( and ). Our gel shift experiments suggest that PUF60 and U2AF65/35
may bind sequentially, rather than simultaneously to the RNA. One possible mechanism is that U2AF65/35
binds initially and recruits PUF60, which subsequently or concomitantly displaces U2AF from the RNA. It is also possible that U2AF is not fully displaced, but that its interaction with the 3′ splice-site is weakened in the presence of PUF60. This change in affinity could reflect an important transition in the spliceosomal complex as splicing proceeds. Although our analysis of the PUF60 complex confirmed the presence of U2AF65
, only two peptides were found by mass spectrometry (), suggesting that interactions between the proteins may be relatively transient.
Spliceosome assembly in the 3′ splice-site region of the intron is very dynamic. Early in the process, interactions between SF1 and the U2AF heterodimer allow for cooperative RNA binding that is important for initial branchpoint sequence recognition 
. An interaction between SF3b155 and U2AF65
replaces the U2AF65
-SF1 interaction and is important for stable U2 snRNP binding to the branchpoint sequence 
binding to the RNA also becomes destabilized during this process 
. In our PUF60 complex () we identified SF3b155 but not SF1. One possible scenario is that SF1 binds cooperatively with U2AF65
, which then recruits PUF60. The arrival of PUF60 could recruit SF3b155 and initiate the replacement of U2AF-SF1 with SF3b155, as well as the stable U2 snRNP association, accompanied by destabilization of U2AF65/35
binding. Many alternatives can also be envisioned, including the possibility that PUF60 functionally overlaps with SF1 in the recruitment of U2AF65
to the RNA. Such a mechanism could explain why SF1 does not appear to be essential for splicing in cells 
. More detailed experiments aimed at understanding the mechanistic interplay of PUF60 and U2AF65
are required to better define the interactions of these proteins and the precise role of PUF60 in 3′ splice site selection.
The isolated PUF60 complex ( and ) offers some clues to the role of PUF60 in splicing. This complex is composed mainly of splicing factors with functions in early spliceosome assembly, including SR proteins and U1 and U2 snRNP components, as well as a putative human homolog of PRP5, an RNA-dependent ATPase. Interestingly, yeast PRP5 forms a bridge between U1 and U2 snRNPs during pre-spliceosome assembly, an association that appears to be important for U2 snRNP interaction with the pre-mRNA 
. The presence of these particular components in the PUF60 complex further suggests that PUF60 is involved in early spliceosome assembly, perhaps by helping to recruit or stabilize U2 snRNP binding. Collectively, our results suggest that PUF60 associates with a subset of splicing factors that likely reflect its function in splicing during early events of the reaction.
PUF60 and U2AF65 as a Functional Class of Splicing Factors
One model for the mechanism of PUF60 in splicing supported by our results is that PUF60 and U2AF65
have distinct functions in splicing, but these functions may be partially interchangeable or conditionally dispensable. Although it has been generally accepted that U2AF65
is required for pre-mRNA splicing in metazoans (
and reviewed in 
), we demonstrate that splicing in vitro
can occur in the absence of U2AF65/35
(). Under these conditions, PUF60 is required in the extract to sustain splicing. At the same time, these two proteins act cooperatively to stimulate splicing at a level more than 5-fold greater than expected if the activities of PUF60 and U2AF65
were independent of each other. Thus, although splicing can occur in the absence of either protein, it is much more efficient when both are present.
Splicing was previously shown to occur in the absence of U2AF65
under certain experimental conditions. One report provides evidence that when nuclear extract is prepared from cells infected with adenovirus, in vitro
splicing of some substrates is dependent on the presence of U2AF65 
; however, splicing of other substrates can occur in the absence of U2AF65
. Another study suggesting the dispensability of U2AF65
reported that in vitro
splicing can be restored in U2AF-depleted extract by the addition of an excess of the SR protein SC35 
Our results raise the possibility that PUF60 and U2AF65
may belong to a family of factors that can modulate splicing based on substrate-specific, early recognition of distinct 3′ splice sites. Another protein, HCC1, which is structurally related to PUF60 and U2AF65 
may be another factor involved in this mode of regulation. HCC1 has been shown to interact with splicing factors such as SRp54 
and SRrp53 
and is found in the spliceosome (reviewed in 
). Related to the notion that these proteins may represent a class of regulatory factors, a recent RNAi screen in Drosophila
aimed at identifying splicing regulators found that knockout of hfp
, the PUF60 ortholog, influences alternative splicing of a partially overlapping set of substrates, compared to knockout of HCC1 and U2AF50, the U2AF65
Regulation of Alternative Splicing by PUF60 and U2AF65
If PUF60 and U2AF65 can indeed modulate splicing based on differential splice-site strengths and/or different requirements for their activities in the splicing of particular introns, then regulation of individual pathways via control of PUF60 and U2AF65 expression levels, localization, or activities could play an important role in alternative splicing and tissue-specific splicing. Indeed, we have identified several alternative splicing events that are altered by such fluctuations in cells ( and ).
Our observation that PUF60 depletion from HeLa cells shifts APP and BIN1 processing to favor brain-specific splicing () suggests that PUF60 may be one factor that helps determine non-neuronal splicing patterns, and the relatively low levels of PUF60 in neuronal cell lines may be partially responsible for the observed skipping of exon exons 7 and 8 in these cells. More extensive experiments testing the effect of PUF60 over-expression in neuronally-derived cells are required to confirm this activity. In this first documented role of PUF60 in alternative splicing, the protein appears to influence splicing of some regulated exons. Interestingly, U2AF65 had different effects on APP and BIN1 splicing compared to PUF60.
The regulation of splicing by PUF60 and U2AF65 appears to be complex, and at this point not readily predictable. We have identified splicing events that are only altered by U2AF65, others that are altered in a similar fashion by both proteins, and still other transcripts that are apparently unaffected by the depletion of either protein. There are no obvious sequence patterns in the 3′ splice sites of these transcripts that correlate with PUF60 or U2AF65 sensitivity. Identifying such features will be an important goal in understanding the mechanism of regulation by these splicing factors.
For some transcripts, such as BIN1
, the depletion of U2AF65
) or both U2AF65
and PUF60 (SMN2
) results in an increase in exon inclusion. These results argue that as yet unknown features of a splice site dictate its dependence on one or the other protein. For example, in the case of SMN2
, the predominant skipping of exon 7 has been attributed to the disruption of a splicing enhancer in exon 7 
. This splicing enhancer is intact in the SMN1
gene—a paralog of SMN2
—whose transcripts efficiently include exon 7. Exonic splicing enhancers recruit U2AF65
to upstream 3′ splice sites 
. Thus, SMN2
exon 7 skipping may be a direct consequence of inefficient U2AF65
binding. It is possible that the depletion of U2AF65
weakens the recognition of the exon 8 3′ splice site, but has little effect on exon 7 splicing, which is already compromised in its ability to recruit U2AF65
. Thus, the strength of the exon 7 and exon 8 3′ splice sites may be equalized by U2AF65
or PUF60 depletion, and thus these sites become more competitive for pairing with the 5′ splice site of exon 6. The outcome of this shift in splice-site recognition would predict an increase in exon 7 inclusion, as observed in . Indeed, masking the exon 8 3′ splice site with an antisense oligonucleotide results in more efficient exon 7 inclusion 
Further evidence of a role of PUF60 in alternative splicing in vivo
comes from hypomorphic mutants of the Drosophila
ortholog of PUF60, Half pint (Hfp), which exhibit alterations in developmentally regulated alternative splicing 
. Knockout of Hfp 
or the PUF60 ortholog in C. elegans 
is embryonic lethal, indicating an essential role for the protein in invertebrate development.
Models for Splicing Regulation by PUF60 and U2AF65
The knockdown of PUF60 and U2AF65 in cells results in changes in certain alternative splicing patterns. In cells in which PUF60 and/or U2AF65 levels become limiting, two possible scenarios can be envisioned for the mechanism of splicing regulation. First, the two proteins may substitute for each other in the splicing reaction, similar to our observations in vitro. This could mean that one can take over the function of the other, or that the activity of one can compensate for loss of the activity of other. In either case, recognition of individual splice sites may be affected differentially by the loss of one or the other protein, depending on the relative strength of a splice site's interaction with, or dependence on, PUF60 or U2AF65. This model involving differential dependence of individual 3′ splice sites on PUF60 and U2AF65 predicts an alteration in splicing patterns when one of the proteins becomes limiting. Alternatively, the lower levels of PUF60 and U2AF65 may result in a limited number of fully functional spliceosomes. Under such limiting conditions, stronger splice sites are predicted to out-compete weaker ones for binding by splicing factors, and thereby alter splicing patterns. Differential recognition may be based on the strength of interaction of the binding sites with splicing components, or perhaps on the presence of specific sequences that recruit PUF60 or U2AF65 to the intron. Overall, our results suggest that 3′ splice-site strength may be defined in part by the relative dependence on the cooperativity between PUF60 and U2AF for recognition.