Higher order structure of Balbiani ring premessenger RNP particles depends on certain RNase A sensitive sites

Specific premessenger ribonucleoprotein (RNP) particles, the Balbiani ring (BR) granules from Chironomus tentans salivary glands, were treated with RNase A to study the effect of RNA strand breaks on the higher order structure of the particles. Isolated, radioactively labeled BR granules, known to sediment at 300 S, were digested with RNase A and centrifuged in sucrose gradients. The fractionated particles were subsequently analyzed using electron microscopy and caesium chloride centrifugation. At a low RNase concentration, most of the 300 S particles disintegrated completely, and no metastable degradation products were observed. At intermediate RNase concentrations, no 300 S particles were left, but a minor fraction of the BR granules had unfolded and sedimented at 160 S. These granules could represent particles modified during the RNase treatment or represent a more slowly degrading subfraction of the particles. At a high RNase concentration, no RNP particles at all remained in the gradient. The rapid disintegration of the majority of the BR granules was investigated further by electrophoretic analysis of RNA in the remaining particles. During the RNase treatment BR granules, still sedimenting at 300 S, accumulated strand breaks; in fact, as many as 50 to 100 nicks in the 37 kb RNA could be tolerated. It was concluded from RNA analyses that the disintegration of the BR granules was not dependent on any single nick in the RNA, nor on the accumulation of a certain number of nicks, but rather on one or a few critical strand breaks. We propose that there are organizing sequences essential for particle integrity; once these sequences are nicked, the premessenger RNP particles are rapidly and completely degraded.