In vivo selection of RNA sequences involved in nucleocytoplasmic RNA trafficking

We used Xenopus oocytes and microinjection techniques to select for RNAs containing nuclear localization signals. A 20 nucleotide long randomized sequence was inserted into a U1-like carrier RNA giving a pool of over 10(12) possible RNA sequences. RNAs that were found in the nucleus 20 hours after nuclear or cytoplasmic injection were amplified and used for the next round of selection and amplification. After 12 rounds, the pools were highly enriched for RNAs that remained in, or were transported into, the nucleus. When individual RNAs of these pools were cloned and sequenced two different types of nuclear localization signals were identified. Some RNAs contain a binding site (whose consensus is AAUUUUUGG) for the Sm proteins common to spliceosomal snRNPs; the 5' caps of these RNAs are hypermethylated during nuclear localization and the RNAs follow the transport pathway of snRNAs. Other molecules could be folded to make long duplexes at their 5' ends; nuclear localization of these RNAs is independent of the 5' caps, and transport from the cytoplasm resembles the pathway for protein import.