Red cell ghost-mediated microinjection of RNA into HeLa cells. II. Cellular translation of protamine mRNA; post-translational modifications and nuclear binding of newly-synthesized protamine.
Red cell ghosts loaded with protamine messenger RNA (pmRNA) were fused to HeLa cells using polyethylene glycol, as a means of introducing the mRNA into heterologous cells. The recipient cells were capable of translating the RNA into the three protamine polypeptides, which may be resolved as three peaks (CI, CII, and CIII) by cation exchange chromatography. The synthesis of components CII and CIII was easily observed with possible traces of CI as well. The HeLa cells also phosphorylated CII after synthesis. However, this phosphorylation did not occur with CIII. In addition, CII but not CIII localized in the nucleus of the HeLa cells after synthesis. Thus, a correlation of post-translational modification with nuclear entry was observed. Localization in the nucleus, however, was not accompanied by the same tight binding of protamine to chromatin as is seen in the homologous trout testis spermatid cells. In the spermatid cells, protamine elutes from chromatin at a salt concentration of 1.2 M NaCl. In contrast, in the HeLa cells, the newly synthesized CII which had entered the nucleus, could be eluted with 0.6 M NaCl. Thus, the tight binding of protamine to chromatin in trout testis may require a series of concomitant developmental events, such as core histone hyper-acetylation (Christensen, M E & Dixon, G-H. In press) [17], which would be lacking in the HeLa cells.
D002451:Cell Compartmentation; D004910:Erythrocyte Membrane; D004912:Erythrocytes; D006367:HeLa Cells; D006801:Humans; D008845:Microinjections / Q000379:methods*; D011479:Protamines / Q000096:biosynthesis*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000008:administration & dosage* / Q000378:metabolism; D013997:Time Factors
D011479:Protamines; D012333:RNA, Messenger