hnRNP proteins are proposed to play essential roles in pre mRNA processing and nuclear export. The highly conserved process of mRNA export is perhaps best understood in the yeast S. cerevisiae where many factors have been defined, one of which is the major mRNA-binding protein Npl3. In an effort to understand how Npl3 is involved in mRNA export, we designed a genetic screen to identify mutations that block Npl3 export, and we successfully identified known mRNA export factors. As a result of this screen, we also found that mutation of TBP could affect both Npl3 and mRNA export. Next, we showed that Npl3 exists in a complex with RNA Pol II and that Npl3 association with genes is transcription dependent. Finally, we have shown that another mRNA export factor, Yra1, is also cotranscriptionally recruited at a later step of transcription. Taken together, these results suggest that the process of mRNA nuclear export begins at the level of transcription.
SPT15 and NPL3 interact to promote mRNA export
The mRNA that is produced in spt15-ts1 npl3-27 double mutant cells is not efficiently exported from the nucleus. One possibility is that reduced transcription levels give rise to this defect. In spt15-ts1 cells, TBP is reduced in its ability to bind promoter DNA at the nonpermissive temperature, thereby compromising its role in transcription initiation and recruitment of the preinitiation complex to the promoter (Cormack and Struhl 1992
). However, the reduced level of transcription cannot be the sole cause of the mRNA export defect because transcription levels are equally reduced in the spt15-ts1 single mutant and spt15-ts1 npl3-27 double mutant, and npl3-27 alone does not affect mRNA levels. Furthermore, when npl3-27 is combined with mutations in Pol II that reduce transcription levels, mRNA export is not affected. In addition, we found that Npl3-27 and mRNA export is also affected in two other spt15 ts
mutants. Therefore, there is a specific genetic interaction between NPL3 and SPT15 to promote mRNA export that may not be entirely dependent on transcription levels.
Another possibility is that in spt15-ts1 npl3-27 cells, decreased activity of TBP combined with the smaller population of Npl3-27 in the nucleus causes an accumulation of export incompetent mRNAs. This hypothesis assumes that npl3-27 is a loss-of-function mutation, and thus we would expect that expression of wild-type NPL3 would rescue the mRNA export defect of spt15-ts1 npl3-27 cells; however, we have found that this is not the case (E. Lei and P. Silver, unpubl.). Moreover, we have found that npl3-27 exerts a dominant effect over wild-type NPL3 in spt15-ts1 cells to cause an mRNA export defect. Therefore, npl3-27 does not appear to be a simple loss-of-function mutation, suggesting that an alteration of Npl3-27 function with respect to wild type is important for the synthetic mRNA export defect when combined with spt15 mutations.
Although Npl3-27 clearly displays altered localization compared to wild type, the precise nature of the Npl3-27 mutant is unclear. The point mutation E309K lies in a region that contains the NLS. It has been proposed that Npl3 binds to mRNA in the nucleus, remains associated throughout export, and releases from the RNA during reimport (Pemberton et al. 1997). Although the assay is not quantitative, our UV-crosslinking studies consistently reveal that a larger population of Npl3-27 than wild-type Npl3 may exist bound to mRNA. Therefore, cytoplasmic accumulation of Npl3-27 may be tied to an inability to release RNA as efficiently as wild type. Changes in the behavior of Npl3-27 with respect to RNA binding could affect many aspects of mRNA export such as loading of Npl3-27 onto the nascent transcript, packaging of the RNP, and interaction of the RNP with transport factors. We were unable to test the behavior of Npl3-27 by chromatin immunoprecipitation because of the inability to immunoprecipitate the mutant protein (data not shown). Further studies of Npl3-27 function will shed light on the intriguing genetic interaction between NPL3 and SPT15.
Recruitment of Npl3 to transcribing genes
Our results show that Npl3 is recruited to genes in a transcription-dependent manner. Promoter DNA found in the chromatin immunoprecipitations, albeit at lower levels than the coding region, suggest that the earliest step of Npl3 recruitment is at the site of transcription initiation. Although the method of chromatin immunoprecipitation has limited resolution, we were able to see differences between the footprints of Npl3 and Yra1, which associates strongly with the 3' ends of coding sequences, suggesting that Npl3 promoter association is significant. We predicted that there might be a physical interaction between Npl3 and some component of the transcription machinery. Although attempts to coimmunoprecipitate Npl3 with TBP and other transcription factors yielded negative results, we were able to detect an interaction of Npl3 with RNA Pol II, indicating that Npl3 physically interacts with the transcription machinery.
Pol II undergoes cycles of phosphorylation and dephosphorylation at its CTD, and this cycle coincides with Pol II activity (Hirose and Manley 2000). Pol II is hypophosphorylated while engaging in initiation and hyperphosphorylated during elongation. Furthermore, multiple elongation and mRNA processing factors are recruited by the Pol II CTD in a manner that is dependent on the state of CTD phosphorylation. We detected Pol II using an antibody that recognizes the unphosphorylated CTD (8WG16) and not with lower affinity antibodies directed to two different phosphorylation sites in the CTD (H14 and H5). Although elongating Pol II may be partially dephosphorylated in these experiments, these results are consistent with the possibility that Npl3 is recruited by unphosphorylated Pol II in the preinitiation complex and transferred to the nascent RNA at the start of elongation (Fig. 7). Alternately, Npl3 may be recruited to elongating polymerase soon after transcription initiation and then transferred to RNA. Moreover, Npl3 may be further recruited by cooperative self-association or by the RNA itself.
To address whether RNA may be necessary for Npl3 recruitment, we performed chromatin immunoprecipitation after extensive RNase A treatment and obtained similar results as described previously, indicating that Npl3 is not associated with chromatin entirely through RNA contacts (E. Lei and P. Silver, unpubl.). However, it should be noted that RNase A treatment cannot be performed before crosslinking; therefore, it is impossible to rule out that Npl3 association relies on contacts that are mediated by large RNA/protein complexes. Therefore, it remains a possibility that the growing nascent transcript further recruits Npl3. Future studies will determine how RNA, Pol II CTD phosphorylation, and other transcription factors affect Npl3 recruitment.
Another mRNA export factor, Yra1, is cotranscriptionally recruited
The mRNA export factors Aly/REF in metazoans and Yra1 in yeast have been proposed to mark fully processed mRNAs for nuclear export (Luo and Reed 1999; Sträßer and Hurt 2000). Although Aly/REF has been shown to preferentially bind to spliced and not unspliced RNAs, there is evidence that Aly/REF mediates the nuclear export mRNAs regardless of whether they once contained an intron (Luo and Reed 1999; Rodrigues et al. 2001). Our chromatin immunoprecipitation experiments, which provide a snapshot of the in vivo association of Yra1 with PMA1 and GAL10, two genes that do not contain introns, are in agreement with Yra1 being a general export factor. Furthermore, Yra1 has been shown to interact directly with the mRNA export factor Mex67, which localizes primarily to nuclear pores, suggesting that Yra1 can function to bridge the formation of the RNP with the actual translocation machinery (Sträßer and Hurt 2000). If this is indeed the case, our results imply that full maturation of the RNP can occur cotranscriptionally. Mounting evidence for cotranscriptional recruitment of multiple factors involved in pre-mRNA processing supports this hypothesis.
Transcription coordinates assembly of the export-competent RNP
NPL3 lies at the center of an increasingly complex network of genes. One such gene is HRP1, which encodes another abundant hnRNP (Henry et al. 1996). Hrp1 is involved in 3' processing and nonsense mediated decay and recently has been shown to be associated with transcribed genes similar to the pattern exhibited by Npl3 (Kessler et al. 1997; Gonzalez et al. 2000; Komarnitsky et al. 2000). Like Npl3, Hrp1 shuttles between the nucleus and cytoplasm dependent on ongoing transcription (Shen et al. 1998). Furthermore, NPL3 genetically and physically interacts with the CBC gene CBP80 in that combinations of mutant alleles of npl3 and cbp80 display synthetic lethal interactions, and Npl3 is strongly associated with the CBC proteins Cbp80 and Cbp20 in an RNA-dependent manner (Shen et al. 2000). We have found that CBP80 is not required for Npl3 recruitment to transcribing genes (E. Lei and P. Silver, unpubl.). Although CBC has not been shown to bind to RNA cotranscriptionally in yeast, this result raises the possibility that Npl3 promotes CBC recruitment to nascent RNAs.
In combination with previously published reports, our data show that mRNA export and processing factors are cotranscriptionally recruited to perform their function in maturation of the RNA for nuclear export. This mechanism may explain early observations in which mRNA export in Xenopus was stimulated by injection of promoter DNA containing an intact consensus TATA-box (de la Pena and Zasloff 1987). Additionally, identification of ptr6+, which encodes a putative TBP-associated factor (TAF), in a screen for mRNA export genes in S. pombe suggests that additional transcription factors may be involved in this process (Shibuya et al. 1999). In sum, we have shown that chromatin immunoprecipitation is a powerful technique that can be used to order the steps of recruitment of proteins to RNA, an outstanding question in the field of mRNA processing and export. Our results suggest that Npl3 recruitment is an early event, possibly occurring at transcription initiation and that Yra1 recruitment is a later event, perhaps signaling the complete maturation of the RNP for export (Fig. 7). Future studies will further understanding of RNP assembly.
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