Human tRNA^Secassociates with HeLa membranes, cell lipid liposomes, and synthetic lipid bilayers

Abstract

We have shown previously that simple RNA structures bind pure phospholipid liposomes. However, binding of bona fide cellular RNAs under physiological ionic conditions is shown here for the first time. Human tRNA Sec contains a hydrophobic anticodon-loop modification: N 6 -isopentenyladenosine (i 6 A) adjacent to its anticodon. Using a highly specific double-probe hybridization assay, we show mature human tRNA Sec specifically retained in HeLa intermediate-density membranes. Further, isolated human tRNA Sec rebinds to liposomes from isolated HeLa membrane lipids, to a much greater extent than an unmodified tRNA Sec transcript. To better define this affinity, experiments with pure lipids show that liposomes forming rafts or including positively charged sphingosine, or particularly both together, exhibit increased tRNA Sec binding. Thus tRNA Sec residence on membranes is determined by several factors, such as hydrophobic modification (likely isopentenylation of tRNA Sec ), lipid structure (particularly lipid rafts), or sphingosine at a physiological concentration in rafted membranes. From prior work, RNA structure and ionic conditions also appear important. tRNA Sec dissociation from HeLa liposomes implies a mean membrane residence of 7.6 min at 24°C (t 1 ⁄ 2 = 5.3 min). Clearly RNA with a 5-carbon hydrophobic modification binds HeLa membranes, probably favoring raft domains containing specific lipids, for times sufficient to alter biological fates.

Transfer RNA Bound to MnmH Protein Is Enriched with Geranylated tRNA – A Possible Intermediate in Its Selenation?

“…A human tRNA Sec containing the prenylated modified nucleoside 6-isopentenyl-adenosine (i 6 A) has an anticodon loop that is highly hydrophobic. Such tRNA is retained in the hydrophobic HeLa cell membrane demonstrating that some tRNAs may be located in another cellular compartment than the cytoplasm [ 38 ]. The prenylated geranyl group of (c)mnm 5 ges 2 U, which is also highly hydrophobic, might mediate insertion of a geranylated tRNA into the hydrophobic lipid bilayer similar to the insertion of tRNA Sec into the human membrane.…”

Chemical Deprenylation of N6‐Isopentenyladenosine (i6A) RNA

“…An N 6 -prenyl adenosine phosphoramidite was prepared ( Figure S4) for the solidphase synthesis of RNA oligo 5'-AGGCUUCAi 6 A-3' (Figures 2 a and S5,6), a motif mimicking the anticodon loop of human tRNA Sec . [12] The deprenylation was found dosedependent (Figure 2 a,b, Figure S7 and Table S4). The HPLC curves obtained from cation 2-induced i 6 A RNA deprenylation revealed the gradual disappearance of the starting oligo with the increase of cation 2 (up to 40 equiv) and the generation of a new peak, which was later purified and characterized by ESI mass spectrometry.…”

Mechanisms of RNA loading into exosomes

“…The presence of sphingosine increases affinity of tRNA to membrane rafts [50]. Since ceramide in the MVB limiting membrane is converted to sphingosine through the action of ceramidase [59] and ceramide is produced in the raft‐like region, sphingosine molecules in the limiting membrane likely increase the affinity of RNAs to this membrane.…”

“…There is another RNA property, besides the membrane binding motif, which can affect the affinity of an RNA molecule to the membrane: its hydrophobic modification. Some mammalian miRNAs can be methylated [75] and tRNAs can have several hydrophobic modifications, including isopentenylation which increases tRNA Sec affinity to the raft‐like region of the membrane [50]. Regulatory small RNAs with miRNA‐like features that are derived from cleavage of tRNA Sec are likely hydrophobically modified because some reported reads overlap with the isopentenylated nucleotide 37 [76].…”