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CELL BIOLOGY Case Made for Nuclear Export LicenseStudy Shows Link Between Splicing and Exporting RNA A link between two fundamental mechanisms of the cell has been discovered by a team of researchers headed by Robin Reed, HMS professor of cell biology. Writing in the Sept. 21 Nature, Reed and her colleagues show for the first time that the protein Aly connects the splicing of pre-messenger RNA in the cell nucleus with the export of spliced messenger RNA to the cytoplasm. Aly is the counterpart in mammals of a known nuclear export factor in yeast, Yra1p, raising the likelihood that the mechanism for mRNA export is highly conserved from single-celled yeast to mammals, including humans, according to Reed.
The protein Aly seems to bridge the splicing and export processes of RNA: From top to bottom, the spliceosome assembles to edit pre-messenger RNA by cutting out the nonreading introns and splicing together the reading exons. Stored in nuclear speckles with other spliceosomal parts, Aly is recruited, becomes tightly associated with the now spliced mRNP, and is exported with the mRNP into the cytoplasm. Then Aly shuttles back to the speckles. The Protein's PathWorking with graduate student Zhaolan Zhou and postdoctoral fellow Ming-juan Luo, Reed found that Aly is recruited to the spliceosome, a dynamic complex of proteins in the nucleus that splices pre-messenger RNA, the transcribed form of DNA. The spliceosome cuts out the introns, the long "junk," or noncoding, portions of RNA, and splices together the exons, the working instructions, into a messenger RNA protein complex called the mRNP. The researchers found that Aly tightly associates with this complex. The introns are retained in the nucleus and degraded. The exons contained in the mRNP are exported to the cytoplasm and translated into protein. A direct link between the spliceosome and the nuclear export mechanism had never been found until Aly."It's recruited to the spliceosome and tightly associated with the mRNP, and Aly is also the first example of a protein that specifically stimulates the export of mRNA. This bridges the two different processes," said Reed, "so you have splicing on the one hand and export on the other and then this factor, Aly, links these two together because it has properties common to both." Conflicting TheoriesThe linkage of splicing and exporting has been controversial, Reed acknowledged. Earlier theories on how mRNA is exported centered on a set of nuclear proteins called hnRNPs, which are abundant in the mammalian nucleus and bind to unspliced pre-messenger mRNA. Some hnRNP proteins were shown to shuttle back and forth from the nucleus to the cytoplasm and were proposed as key players in exporting mRNA. However, the hnRNP model of export concerned Reed because the hnRNPs also associate with mutant pre-messenger RNAs that fail to enter the spliceosome assembly pathway; if hnRNPs were central to export they might also export the mutant RNAs, which could interfere with normal gene expression.
Robin Reed (left) takes a break from RNA processing with Ming-juan Luo and Zhaolan Zhou (while Aly the Alligator lurks behind them). Photo by Steve Gilbert Reed, who has been studying the splicing pathway for 10 years, had already demonstrated that the spliceosome essentially excludes hnRNPs as it goes about its work. Since splicing is upstream from export, she began to wonder if the spliceosome was somehow marking spliced mRNA as distinct from the RNA packaged by hnRNPs, and thus safe for export. Then Reed and Luo found evidence for this suspected link. In a paper published in PNAS last December, they demonstrated that splicing itself was a requirement for efficient mRNA export and that a specific mRNP assembled on the spliced mRNA to aid efficient export. Reed believed that this mRNP would act as a kind of license, stamping mRNA from the spliceosome pathway as ready for export. "We wanted to ask the question, when it goes through the splicing pathway and assembles into the mRNP, what marks it for export?" At the time, no factors had been found in mammals to be essential for mRNA export, Reed recalled. Then she learned that Ed Hurt of the University of Heidelberg had identified Yra1p as an essential nuclear export factor in yeast. He also showed that Yra1p had a mammalian homologue, Aly. Could Aly be the stamp on the mRNP that promotes export? Strikingly, Reed's experiment found that Aly is specifically recruited to the spliceosome and then tightly associated with the mRNP. "It's what we predicted. Splicing does lead to formation of an mRNP that recruits the export machinery, and a key factor that's involved is Aly." They also found that another protein, Tap, was present in low levels in the mRNP. Just as Aly has an essential yeast homologue in Yra1p, Tap has a counterpart called Mex67p that is essential for export in yeast. This strengthened the case that the linking of splicing and export is highly conserved across species. A Well-behaved ProteinImportantly, Aly does not associate with mRNPs that are already lacking introns or with hnRNP complexes. This makes Aly an ideal export license, said Reed. "The interesting biological reason for linking splicing to export is the opposite of that of the model where hnRNPs are involved in export. I think that hnRNPs may be involved in a kind of proofreading mechanism—mutant pre-mRNAs that can't go through the splicing machinery are bound only to hnRNP proteins. By contrast, normal pre-mRNAs are spliced and therefore associate with the nuclear export factor Aly. Thus, the spliced RNA is basically stamped by Aly and other marker proteins that say, 'This should get out.' Whereas if it's a mutant RNA or an intron, it may be bound in hnRNP complexes and retained in the nucleus and degraded."Aly is not an hnRNP protein, said Reed. It is not found in the hnRNP complex. It does not colocalize with hnRNPs in the nucleus. Instead, Aly is found in nuclear "speckles," 20 to 50 localized regions in the cell nucleus that hold the molecular parts for the spliceosome. Speckles contain 50 or so special spliceosomal proteins and five snRNPs (pronounced "snurps"), small RNA protein complexes. The speckles are exactly where you would expect to find an export factor that links splicing to export, said Reed "We found that not only is Aly present in speckles and associates with the mRNP, but we went on to show that it is able to stimulate export of spliced mRNA. This is the first example of any protein that does this in metazoans." —John Fleischman |
