The Transacting Factor CBF-A/Hnrnpab Binds to the A2RE/RTS Element of Protamine 2 mRNA and Contributes to Its Translational Regulation during Mouse Spermatogenesis
Figure 6
The translationally active Prm2 mRNA associates with the p42 CBF-A isoform.
(A–B) Fractionation of adult mouse testicular lysates on a 15–50% continuous sucrose gradient in the absence (A) or presence (B) of EDTA. Fractions were analyzed on Northern blots for the Prm2 mRNA and on immunoblots for CBF-A (SAK22) and hnRNP A2. (C–D) The Prm2 mRNA is translationally inhibited in postnatal day −28 and −30 mouse testis. (C) Developmental expression of Prm2 mRNA in mouse testes. Total RNA was collected from mouse testis of 20, 28, 32 postnatal day of age (20–32 d), and analyzed by RT-PCR. RT, reverse transcriptase. (D) Developmental expression of PRM2 and CBF-A in mouse testes. Cytoplasmic testicular extracts from 20, 28, 30, 32 d mice were analyzed on immunoblots for PRM2, CBF-A (SAK22) and tubulin. (E) RIPs from adult and 30 d mouse testicular extracts. Cytoplasmic fractions were incubated with SAK22, ICCI or control anti-mouse IgGs. Immunoprecipitated fractions were analyzed on immunoblots for CBF-A (SAK22) and hnRNP A2, and by RT-PCR with primers amplifying the Prm2, α-tubulin or clusterin cDNAs. (F–G) The CBF-A p42 isoform associates with the 5′ mRNA cap complex. Cytoplasmic fraction from adult mouse testis was incubated with 7-methyl-GTP (m7GTP)-Sepharose or protein G-Sepharose (control). Bound proteins were analyzed on immunoblots for CBF-A (SAK22), hnRNP A2 and eIF4E and α-tubulin. (F) Lanes 1, 2 respectively 2% and 1% input. (G) Lane 1 and 2 are 2% input. Where indicated, testis lysates were pre-incubated with RNase A prior to incubation with the beads.