3′‐Box‐dependent processing of human pre‐U1 snRNA requires a combination of RNA and protein co‐factors

Using an in vitro system we have recently shown that the 3′ ends of human pre‐snRNAs synthesized by RNA polymerase II are produced by RNA processing directed by the snRNA gene‐specific 3′ box. Towards a complete characterization of this processing reaction we have further investigated the in vitro requirements for proper 3′ end formation of pre‐U1 snRNA. Here we show that the 5′ cap plays a stimulatory role and processing requires creatine phosphate. Our results also indicate that the pre‐U1 processing activity is heat sensitive and that an RNA component is required. In addition, the exact sequence adjacent to the 3′ box influences the position of the pre‐U1 3′ end produced in vitro. Interestingly, the processing extract active for 3′‐box‐dependent processing also contains an activity that converts the 3′ end of RNA containing the U1 Sm protein binding site and the 3′ terminal stem–loop into the mature form.