Sequence-dependent recruitment of SRSF1 and SRSF7 to intronless lncRNA NKILA promotes nuclear export via the TREX/TAP pathway

Abstract

The TREX-TAP pathway is vital for mRNA export. For spliced mRNA, the TREX complex is recruited during splicing; however, for intronless mRNA, recruitment is sequence dependent. However, the export of cytoplasmic long noncoding RNA (lncRNA) is poorly characterized. We report the identification of a cytoplasmic accumulation region (CAR-N) in the intronless lncRNA, NKILA. CAR-N removal led to strong nuclear retention of NKILA, and CAR-N insertion promoted the export of cDNA transcripts. In vitro RNP purification via CAR-N, mass spectrometry, and siRNA screening revealed that SRSF1 and SRSF7 were vital to NKILA export, and identified a cluster of SRSF1/7 binding sites within a 55 nucleotide sequence in CAR-N. Significant nuclear enrichment of NKILA was observed for NKILA lacking CAR-N or the cluster of binding sites in knock-in models. Depletion of TREX-TAP pathway components resulted in strong nuclear retention of NKILA. RNA and protein immunoprecipitation verified that SRSF1/7 were bound to NKILA and interacted with UAP56 and ALYREF. Moreover, NKILA lacking CAR-N was unable to inhibit breast cancer cell migration. We concluded that the binding of SRSF1/7 to clustered motifs in CAR-N facilitated TREX recruitment, promoting the export of NKILA, and confirmed the importance of NKILA localization to its function.

Application of RNA subcellular fraction estimation method to explore RNA localization regulation

“…RBM4 , which is the most enriched RBP in the cytosolic genes, contains a C-terminal alanine-rich domain that potentially mediate RNA export ( Lai et al 2003 ). RNA export function of SRSF1 , another top enriched RBP, is also supported by a few published literatures ( Das and Krainer 2014 ; Khan et al 2021 ). However, the functional roles of RBP in RNA localization, such as RNA export, were still very limited.…”

“…For separation of nuclear and cytoplasmic fractions, the extraction kit (Beyotime China, P0027) was used and followed the same procedures as previous study ( Khan et al 2021 ). Total RNA was used as input material for the RNA sample preparations.…”

Predictive and Prognostic Value of Non-Coding RNA in Breast Cancer

“…A recent study indicated that cytoplasmic accumulation region (CAR-N) removal in NKLA, an intron-less lncRNA, results in significant nuclear restriction and insertion of CAR-N increased cDNA transcript export. Researchers also found that TREX-TAP pathway depletion strongly restricts NKILA to the nucleus and that removal of CAR-N does not prevent breast cancer cell migration [ 28 ]. Previously, Zuckerman et al depleted several proteins that are involved in nuclear export.…”

Predictive and Prognostic Value of Non-coding RNA in Breast Cancer

“…A recent study indicated that cytoplasmic accumulation region (CAR-N) removal in NKLA, an intronless lncRNA, results in significant nuclear restriction and insertion of CAR-N increased cDNA transcript export. Researchers also found that TREX-TAP pathway depletion strongly restricts NKILA to the nucleus and that removal of CAR-N does not prevent breast cancer cell migration [26]. Previously, Zuckerman et al depleted several proteins involved in nuclear export.…”