We previously characterized a nuclear protein from N
, NpUBP1, associated with the nuclear poly(A)+
RNA in vivo, as demonstrated by UV cross-linking experiments performed with intact cells. NpUBP1 has binding specificity for oligouridylates and interacts with U-rich intron and 3′-UTR sequences in vitro. Transfection experiments indicated that the overexpression of NpUBP1 has two independent posttranscriptional effects on reporter mRNAs. It enhances the splicing of suboptimal introns and increases steady-state levels of transcripts, even intronless ones (34
). To gain more insight into how UBP1 functions in pre-mRNA maturation, we have now characterized two A
proteins interacting with NpUBP1 and its A
counterparts. These proteins, UBA1a and UBA2a, are members of two novel families of RRM domain-containing proteins which appear to be expressed only in higher plants. Like NpUBP1, UBA1a and UBA2a are RNA-binding proteins localized in the nucleus. Both proteins interact with 3′UTR sequences and have strong binding preference for oligouridylates in vitro.
Analysis of the effects of UBA1a and UBA2a overexpression on reporter RNA maturation revealed some similarities with but also substantial differences from UBP1. Like that of UBP1, the expression of either of the two new proteins in transfected protoplasts led to increased accumulation of Syn3 and GUS reporter mRNAs, with the effect of UBA2a being significantly more pronounced (Fig. and ). Previous efforts to elucidate the mechanism of the stimulatory effect of UBP1 on mRNA abundance suggested that UBP1 may bind to the 3′-UTR of reporter mRNAs and prevent exonucleolytic degradation from continuing beyond the poly(A) tail. This conclusion was based on the observations that UBP1 efficiently cross-links to the 3′-UTR region in vitro and that its overexpression in protoplasts results in the accumulation of transcripts corresponding to the poly(A)-free form of mRNAs (34
). Northern analysis performed in this work indicated that, in marked contrast to UBP1 overexpression, neither UBA1a nor UBA2a overexpression leads to the appearance of the poly(A)-free mRNA (Fig. ). In light of the results demonstrating various interactions among the three proteins, it is plausible that UBP1, UBA1a, and UBA2a participate in different complexes, with specificity defined by their precise compositions. Overexpression of one of the components could promote the formation of unique complexes with distinct properties. It is possible, for example, that UBA1a and UBA2a interact with the poly(A)-binding protein and, when overexpressed, predominantly stabilize its interaction with poly(A) tails, leading to higher stability of poly(A)+
RNA. In contrast, UBP1 may not contribute to such interactions and may affect mainly the level of the poly(A)-free mRNA. It will be interesting to investigate whether UBA1a and UBA2a interact with the poly(A)-binding protein.
Experiments carried out with yeast and mammalian cells demonstrated that substantial fractions of pre-mRNA transcripts undergo rapid turnover in the nucleus (references 5
and references therein) and that the 3′→5′ exonuclease complex (exosome) is involved in this process (5
). Do the UBP1, UBA2a, and UBA2a proteins regulate the turnover of mRNAs or their precursors in the nuclei of plant cells? Localization studies have shown that UBP1, UBA2a, and UBA2a are nuclear proteins (34
; this work). In addition, it was previously demonstrated that UBP1 is associated with the nuclear poly(A)+
RNA in plant cells in vivo (34
) and that the poly(A)−
RNA accumulating upon the overexpression of UBP1 is localized in the nucleus (63
). Taking these and other established properties of UBP1, UBA1a, and UBA2a into account, it is tempting to propose that all three proteins, either individually or as a complex, interact with different regions of the generally U-rich 3′-UTRs in plant cell nuclei. Such interactions may prevent 3′→5′ exonuclease-mediated degradation from continuing beyond the poly(A) tail and may make the RNAs available for readenylation. It is also possible that the proteins facilitate some steps in nuclear pre-mRNA processing or mRNA export to the cytoplasm. However, it is important to note that we have found no evidence of UBP1 having a stimulatory effect on the 3′-end cleavage or polyadenylation reaction in transfected plant protoplasts (34
). It has also been demonstrated that UBP1 does not shuttle between the nucleus and the cytoplasm in a transcription-dependent manner (63
). However, the possibility that the protein shuttles between these compartments in a process independent of transcription has not been eliminated by our experiments.
It was previously shown that the stimulatory effect of NpUBP1 on mRNA abundance in transfected protoplasts is promoter specific. NpUBP1 increased the level of mRNA when transcription was driven by the CaMV 35S promoter but not the cellular GLB promoter, indicating that NpUBP1 may interact with the transcription machinery at the step of initiation. We have now found that the effect of UBA1a, but not that of UBA2a, also depends on the nature of the promoter driving the transcription of the reporter gene. Hence, the association of both UBP1 and UBA1a with transcripts apparently requires their prior interaction with the transcribing RNA polymerase II (PolII) complex. It is now generally accepted that mRNA transcription and processing are coupled in vivo. Many protein factors participating in various RNA processing reactions in mammalian and yeast cells associate with the CTD domain of the large subunit of PolII at transcription initiation (16
; reviewed in references 3
, and 50
). Notably, the recruitment of some mammalian SR proteins to pre-mRNA was shown to be modulated by the promoter structure. As reported previously, we did not find evidence of an interaction between UBP1 and the CTD, either in vitro or in the yeast two-hybrid system (34
). Likewise, the yeast two-hybrid experiments did not reveal interactions of UBA1a and UBA2a with the A
CTD (unpublished results). Despite these negative results, UBP1 and UBA1a may still associate with the CTD, but the interaction may require the simultaneous presence of UBP1 and UBA1a and possibly also of UBA2a or other proteins. Alternatively, the association of the investigated proteins with the PolII complex may depend upon prior interactions with specific transcription factors, as has been described for the ASF/SF2 splicing factor in mammalian cells (11
In contrast to that of UBP1, the overexpression of UBA1a and UBA2a has no effect on the splicing efficiency of reporter RNAs containing suboptimal introns, such as intron SynGC/ClaU (20
) or intron 1 of the maize actin gene (22
). Hence, the interactions among UBP1, UBA1a, and UBA2a described in this work are apparently related to the functions of these proteins in mRNA accumulation or stabilization but probably not in the splicing-promoting activity of UBP1. Establishing the mechanism by which UBP1 stimulates intron processing will require additional experimentation. However, the structural similarity of UBP1 to the TIA-1 and Nam8p proteins (34
) (see also above) makes it plausible that, by binding to U-rich sequences in introns, UBP1 helps to recruit U1 snRNP or other splicing factors to pre-mRNA.
The previous finding that the overexpression of UBP1 stimulates both pre-mRNA splicing and mRNA accumulation raised the possibility that the effect on splicing is indirect, resulting from increased transcript stability in the nucleus. However, the demonstration that the effect of UBP1 on RNA accumulation is promoter specific and that the effect on splicing is not indicated that the two effects are independent (34
). This conclusion is further supported by data on UBA1a and UBA2a. The expression of either protein in protoplasts stimulated the accumulation of reporter mRNAs but not their splicing. Hence, an effect on RNA accumulation need not necessarily result in an apparent increase in pre-mRNA splicing. It should be noted that the effects of UBP1, UBA1a, and UBA2a on RNA accumulation are clearly not splicing dependent, since they were observed with several different intronless reporter RNAs.
The presence of introns can dramatically increase the levels of expression of mRNAs both in mammals and in plants (4
; reviewed in reference 55
). To compensate for the lack of introns, many natural intronless mRNAs in mammalian cells contain sequence elements that enable efficient intron-independent processing and nuclear export (29
). In A
and probably in other plants, approximately 20% of genes lack introns (58
). Previous observations that UBP1 affects the accumulation of only intronless or otherwise suboptimal reporter pre-mRNAs and that this effect occurs in a promoter-dependent manner indicated that UBP1 plays a role in the expression of certain classes of pre-mRNAs (34
). The identification of UBP1-interacting proteins, UBA1a and UBA2a, which stimulate the accumulation of reporter mRNAs in a manner similar (although not identical) to that of UBP1 suggests that complexes of these proteins regulate pre-mRNA metabolism in plants. Like UBP1 (34
; unpublished results), UBA1a and UBA2a are both members of multiprotein families. Different isoforms of the UBP1, UBA1, and UBA2 proteins may form complexes with related but not identical properties important for the expression of specific classes of mRNAs. In the future, it will be important to inactivate individual variants of UBP1 and its associated proteins and to determine the effect on the expression of intronless genes in A