SINEUP long non-coding RNA acts via PTBP1 and HNRNPK to promote translational initiation assemblies

Abstract

SINEUPs are long non-coding RNAs (lncRNAs) that contain a SINE element, which up-regulate the translation of target mRNA and have been studied in a wide range of applications for biological and therapeutic tools, although the molecular mechanism is unclear. Here, we focused on the kinetic distribution of target mRNAs and SINEUP RNAs by performing co-transfection of expression vectors for these transcripts into human embryonic normal kidney cells (HEK293T/17) to investigate the network of translational regulation. The results showed that co-localization of target mRNAs and SINEUP RNAs in the cytoplasm was one of the key phenomena. We identified PTBP1 and HNRNPK as essential RNA binding proteins. These proteins contributed to SINEUP RNA sub-cellular distribution and to assembly of translational initiation complexes, leading to enhanced target mRNA translation. These findings will promote a better understanding of the mechanisms on the fate of regulatory RNAs implicated in efficient protein translation.

Synthetic in vitro transcribed lncRNAs (SINEUPs) with chemical modifications enhance target mRNA translation

“…We previously found that SINEUP-GFP RNAs localized both in the nucleus and in the cytoplasm when pEGFP-C2 and SINEUP-GFP plasmids were cotransfected [17]. We now found that (m)IVT SINEUP-GFP localized in the cytoplasm regardless of EGFP plasmid transfection (Fig.…”

“…RNA fluorescence in situ hybridization (FISH) was performed as previously described [17]. Briefly, cells were fixed in 4% paraformaldehyde (Wako) and permeabilized in 0.5% Triton X-100 (Sigma-Aldrich) at room temperature for 5 min.…”

“…Here, we successfully developed synthetic, chemically modified IVT (mIVT) SINEUPs that upregulated the translation of target enhanced green fluorescent protein ( EGFP ) mRNA and endogenous target sex‐determining region Y (SRY)‐box 9 ( Sox9 ) mRNA in cultured cells. In addition, mIVT SINEUP RNA successfully upregulated EGFP production in a HeLa extract cell‐free translation system, which contains the SINEUP‐specific RNA‐binding proteins (RBPs) HNRNPK and PTBP1 [17].…”

An NMR-based approach reveals the core structure of the functional domain of SINEUP lncRNAs

“…In fact, our analysis by icSHAPE showed high-enrichment-score protected regions in the T domain, further validating the presence of a single-strand region in this domain in living cells, whereas low-enrichment-score regions in the T domain were preferentially double-stranded as a result of the constraints of the RNAfold software ( Supplementary Figure S5 , see ‘Materials and Methods’ section) ( 38 , 53 ). Thus, our NMR results provide new insights into the RNA regions of inverted SINE B2, and the T domain dynamics may be applicable to binding proteins for SINEUP activity in living cells ( 54 , 55 ). They therefore emphasize the importance of experiment-based structural examination.…”

“…However, challenges with in vitro NMR structural analysis still remain, because RNAs adapt to different regulatory states by structural change across different cellular compartments ( 51 ). Because AS-Uchl1 RNAs and synthetic SINEUPs are intercellular mobile elements, nucleocytoplasmic shuttling happens after transcription ( 10 , 55 ). Therefore, different inverted SINE B2 structures may play central roles in the different regulatory states and in different cellular compartments.…”