The positional, structural, and sequence requirements of theDrosophilaTLS RNA localization element

Abstract

The subcellular localization of mRNAs is a key step in the polarization of cells in organisms from yeast to man. Here, we use a transgenic fly/in situ hybridization assay system to define the positional, structural, and sequence requirements of the TLS, a stem loop RNA sequence element that mediates the subcellular localization of K10 and Orb transcripts in Drosophila oocytes. We find that the TLS is a highly robust and modular element. It mediates efficient RNA localization regardless of sequence context or position within the transcript. Site-specific mutagenesis experiments indicate that the size and shape of the stem and loop regions are critical determinants of TLS activity. Such experiments also identify specific base residues that are important for TLS activity. All such residues map to the stem portion of the structure. Significantly, mutations at these residues interfere with TLS activity only when they alter the stereochemistry of the stem's minor groove. For example, mutation of the A:U base pair at position 3 of the TLS stem to G:C severely reduces TLS activity, while mutation of the same base pair to U:A has no effect. Extensive searches for TLS-like elements in other Drosophila mRNAs using sequence and structural parameters defined by our experiments indicate that the TLS is unique to K10 and Orb mRNAs. This unexpected finding raises important questions as to how the many hundreds of other mRNAs that are known or thought to exhibit K10 and Orb-like localization are localized.

Discovery of functional elements in 12 Drosophila genomes using evolutionary signatures

“…39 UTR structures were also enriched for genes involved in mRNA localization (3 out of 38, P 5 2.7 3 10 24 ), including oo18 RNA-binding protein (orb) and staufen (stau), both of which contain doublestranded RNA-binding domains, are involved in axis specification during oogenesis, and interact with the mRNA of maternal effect protein oskar. The hairpin in orb is known to be important for mRNA transport and localization 71 , whereas the highly similar stau hairpin has not been previously described to our knowledge.…”

Cis-acting determinants of asymmetric, cytoplasmic RNA transport

“…It is not clear, however, whether these proteins play dual roles in splicing or transport, or whether splicing is simply a prerequisite for nuclear export and therefore cytoplasmic localization. In a separate study, insertion of a zipcode (the TLS region from the anteriorly localized Drosophila K10 transcript) near a 59 splice site supported localization but impaired splicing (Cohen et al 2005). Whether this result reflects simple steric interference between the localization and splicing machineries or a more complex mechanistic relationship remains unknown.…”

“…This model has largely proven to be correct in the case of the TLS element (Cohen et al 2005), a zipcode present in both the orb and K10 transcripts of Drosophila (Serano and Cohen 1995). The native zipcode is predicted to fold in a stem-loop consisting of a 17 base pair (bp) stem interrupted by two single-base bulges and an 8 base loop (Cohen et al 2005). Fine-mapping studies showed that increasing loop size or decreasing the length of the stem interfered with transport/localization.…”

“…The only exception was at the fifth position of the stem, where the UÁA / AÁU mutation caused subtle defects in localization. It appears, therefore, that the TLS element is recognized on the basis of hydrogen-bond patterns in the minor groove of the helix, with a small contribution from the major groove of base-pair 5 (Cohen et al 2005).…”

“…Although zipcodes are usually located in 39 untranslated regions (UTRs) of transported messages (e.g., Mowry and Melton 1992;Kim-Ha et al 1993;Gavis et al 1996a;Zhou and King 1996b;Deshler et al 1997;Macdonald and Kerr 1997;Chan et al 1999), in some instances they can mediate localization when placed at the 59 end or even in the middle of a reporter RNA (Cohen et al 2005), although not all zipcodes are position independent . Zipcodes in bud-localized yeast RNAs, in contrast to transported RNAs in metazoans, are generally found in coding regions (Shepard et al 2003), but can function efficiently when located in 39 UTRs of reporter transcripts (e.g., Chartrand et al 1999;Gonzalez et al 1999;Hachet and Ephrussi 2004).…”