Many eukaryotic small RNAs transcribed by RNA polymerase Il as well as by RNA polymerase III are processed on their 3' ends. This processing includes deletion of transcriptionally incorporated nucleotides, addition of nucleotides and in some instances formation of a 2' 3' cyclic phosphate. We have evidence for the trimming of transcriptionally incorporated nucleotides and addition of 1-2 adenylic acid residues on the 3' end of 7SK and SRP small RNAs. Our lab recently obtained evidence for the presence of cyclic phosphates in a subpopulation of human U1, U2, U3, U4, U5, 7SK, SRP and 7SM RNAs. Recently, we also developed an in vitro system capable of adding uridylic acid residues to the 3' end of U6 RNA and also forming the cyclic phosphate structure. This in vitro system will be used for studies on the mechanism 3' end processing of U6, SRP and 7SK small RNAs. We propose to extend these initial studies with the following specific aims: 1. The enzymatic activities necessary for the addition of nucleotides on the 3' end and formation of cyclic phosphate structures in vitro will be purified and characterized. 2. To identify the signals that direct the 3'-end trimming, addition of nucleotides and cyclic phosphate formation in U6 snRNA, mutated U6 snRNAs will be assayed for their ability to direct 3' nucleotide addition and the cyclic phosphate formation in vitro. Similar studies will be carried out subsequently with SRP and 7SK small RNAs. 3. To understand the function of the cyclic phosphate structure, small RNAs will be prepared with and without the cyclic phosphate structures and will be introduced into frog oocytes and/or HeLa cells and parameters like stability, transport across nuclear membrane and incorporation into ribonucleoprotein particles will be investigated.
