Native spliceosomes assemble with pre-mRNA to form supraspliceosomes

Regulation of eukaryotic gene expression is achieved at different levels, which require accurate coordination. Macromolecular assemblies that exist as pre-formed entities can account for such coordination. Processing of pre-mRNA represents one step in this cascade of regulatory events but, moreover, provides explanation for protein versatility. The cellular machine where splicing of pre-mRNA, as well as additional processing events, take place in vivo is termed the supraspliceosome. Here, we show that the supraspliceosome is composed of four active spliceosomes, termed native spliceosomes, connected to each other by the pre-mRNA. Cleavage of pre-mRNA shows that its integrity is essential for the stability of the supraspliceosome. Furthermore, supraspliceosomes can be reconstituted in vitro, from purified native spliceosomes by addition of synthetic pre-mRNAs, providing further support to the supraspliceosome as a preassembled biological complex. The internal setting of the native spliceosomes within the supraspliceosome is most suitable to enable the communication between these structures, which is crucial in order to achieve regulated splicing.     

The poly A polymerase Star-PAP controls 3'-end cleavage by promoting CPSF interaction and specificity toward the pre-mRNA

Star-PAP is a poly (A) polymerase (PAP) that is putatively required for 3'-end cleavage and polyadenylation of a select set of pre-messenger RNAs (mRNAs), including heme oxygenase (HO-1) mRNA. To investigate the underlying mechanism, the cleavage and polyadenylation of pre-mRNA was reconstituted with nuclear lysates. siRNA knockdown of Star-PAP abolished cleavage of HO-1, and this phenotype could be rescued by recombinant Star-PAP but not PAPα. Star-PAP directly associated with cleavage and polyadenylation specificity factor (CPSF) 160 and 73 subunits and also the targeted pre-mRNA. In vitro and in vivo Star-PAP was required for the stable association of CPSF complex to pre-mRNA and then CPSF 73 specifically cleaved the mRNA at the 3'-cleavage site. This mechanism is distinct from canonical PAPα, which is recruited to the cleavage complex by interacting with CPSF 160. The data support a model where Star-PAP binds to the RNA, recruits the CPSF complex to the 3'-end of pre-mRNA and then defines cleavage by CPSF 73 and subsequent polyadenylation of its target mRNAs.     

Identification of new poly(A) polymerase-inhibitory proteins capable of regulating pre-mRNA polyadenylation

The 3' ends of nearly all eukaryotic pre-mRNAs undergo cleavage and polyadenylation, thereby acquiring a poly(A) tail added by the enzyme poly(A) polymerase (PAP). Two well-characterized examples of regulated poly(A) tail addition in the nucleus consist of spliceosomal proteins, either the U1A or U170K proteins, binding to the pre-mRNA and inhibiting PAP via their PAP regulatory domains (PRDs). These two proteins are the only known examples of this type of gene regulation. On the basis of sequence comparisons, it was predicted that many other proteins, including some members of the SR family of splicing proteins, contain functional PRDs. Here we demonstrate that the putative PRDs found in the SR domains of the SR proteins SRP75 and U2AF65, via fusion to a heterologous MS2 RNA binding protein, specifically and efficiently inhibit PAP in vitro and pre-mRNA polyadenylation in vitro and in vivo. A similar region from the SR domain of SRP40 does not exhibit these activities, indicating that this is not a general property of SR domains. We find that the polyadenylation- and PAP-inhibitory activity of a given polypeptide can be accurately predicted based on sequence similarity to known PRDs and can be measured even if the polypeptides' RNA target is unknown. Our results also indicate that PRDs function as part of a network of interactions within the pre-mRNA processing complex and suggest that this type of regulation will be more widespread than previously thought.