MARKED FOR EXPORT
Courtesy of Jens Lykke-Andersen
A post-splicing complex deposited upstream of each exon-exon junction tags mRNAs for nuclear export and mRNA surveillance. The pre-mRNA is coated with hnRNP proteins. The Spliceosome deposits an exon-exon junction complex that promotes nuclear export by interaction with TAP and mRNA surveillance by interaction with hUpf proteins.
Throughout the journey from gene to protein, RNAs are encrusted with proteins that splice and dice sequences and inevitably regulate the processing and features of the final product. These nuclear sequence shufflings are implicated increasingly in mechanisms such as RNA-export to the cytoplasm, and nonsense-mediated decay (NMD), which detects and degrades RNAs with premature termination codons (PTCs). The role of splicing in export, however, remained largely unclear. Perhaps even more puzzling, NMD machinery in the cytoplasm needs to distinguish PTCs from legitimate stop codons. This suggested to some that mRNA can remember where its introns were...
RNA'S QUALITY CONTROL
First observed in the early 1980s in mammals, NMD ensures that only mRNAs with full coding potential are translated into protein. In mammals, if an mRNA has a splicing junction more than 50 nucleotides downstream of the first in-frame stop codon, it becomes targeted for NMD. But how the cell could possibly recognize the difference between the premature termination codons and the real termination codon was pure enigma, says Steitz, because this requires that the cytoplasmic translation machinery know the nuclear processing history of a given mRNA. Therefore, either translation components exist in the nucleus, or somehow presplicing history is communicated to the cytoplasm. And so, researchers speculated that mRNA must somehow remember where its introns were before being spliced.
"We called [it] a marker," says Miles Wilkinson, University of Texas, MD Anderson Cancer Center, Houston, "but we had no idea what it was or whether or not it even existed." He adds that the marker explanation reconciles the disparity: "It didn't require hypothesizing anything weird like nuclear translation."
Lynne Maquat, University of Rochester, NY, and others initially hypothesized that splicing-dependent changes in the proteins accompanying mRNA likely facilitate some sort of molecular memory. A protein would mark the exon-exon junctions in the nucleus, and the mark would be carried to the cytoplasm where it is read by translation machinery. Moore and others described that mark as the EJC. The spliceosome deposits the complex roughly 24 nucleotides upstream of exon-exon junctions in a sequence-independent manner.
In 2000, searching for the hypothesized mark, Lykke-Andersen and Steitz described Upf2 and Upf3, human homologs of yeast NMD proteins.2 They then created an artificial mark by tethering the factors onto mRNA downstream of a premature termination codon, which resulted in NMD. "We were so lucky that it mimicked an intron and had that effect," says Lykke-Andersen. Izaurralde comments, "It was the first time to show that tethered proteins could recruit all the necessary machinery to degrade mRNA."
Even though all organisms examined except bacteria manifest NMD, neither the mechanism nor the factors are conserved, says Maquat. "And, as far as I can tell, the role of the EJC in NMD is evident only in mammals."
THE EJC SPRINGBOARD
At the same time the
Courtesy of Elisa Izaurralde
In this assay, proteins of interest are expressed as fusions with the coat protein of bacteriophage MS2. The reporter gene harbors high affinity MS2-binding sites in the 3' UTR. When the stop codon is located at least 50 nucleotides upstream of these biniding sites, NMD factors (e.g., EJC proteins such as Y14) recruit additional components of the NMD machinery and the reporter mRNA is degraded.
This work also confirmed a connection between splicing and mRNA export, at least for very short pre-mRNAs containing no poly-A tails that are injected into
REF: EXIT ASSISTANT
That same year, Izaurralde's group published the third Hot Paper describing REF's role in mRNA export. Their findings suggest that both spliced and unspliced (intronless) mRNAs use common export factors.4 This notion went against the prevailing model and published work stating that spliced mRNA exited the nucleus more efficiently. The key to this research is that it tested intronless mRNAs. "We show that it doesn't matter if there is splicing or not. REF was able to bind TAP and promote export of even unspliced RNA," says Izaurralde. Moore's work shows that shorter RNAs are inefficiently exported unless they carry an EJC.3 Izaurralde determined that this effect becomes less pronounced with longer RNA sequences.4 The probability for REF and other mRNA export adaptors to bind to mRNAs independent of splicing is likely to increase with RNA length, providing an explanation for this length effect, says Moore.
The paper might be highly cited for many reasons, says Izaurralde. The REF proteins are part of the newly discovered EJC on which many groups are working. But the most likely reason, she says, is that the original model was extremely popular, and now most people cite the Hot Paper for its suggestion that splicing is not required for mRNA export, although it may enhance its efficiency.
The idea that splicing has so many effects on downstream mRNA metabolism has not generally been appreciated until now. Once studied in isolation, many disciplines now converge. "There were the splicers, the transcriptors, the translators," says Moore. "We didn't talk to each other as much before, but now we are. That's very exciting."
Linda B. Schultz
For this article
Data derived from the Science Watch/Hot Papers database and the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age.
J. Lykke-Andersen et al., "Human Upf proteins target an mRNA for nonsense-mediated decay when bound downstream of a termination codon,"
H. Le Hir et al., "The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay,"
J. Rodrigues et al., "REF proteins mediate the export of spliced and unspliced mRNAs from the nucleus,"