Subnuclear Localization and Dynamics of the Pre-mRNA 3′ End Processing Factor Mammalian Cleavage Factor I 68-kDa Subunit

Abstract

Mammalian cleavage factor I (CF I m ) is an essential factor that is required for the first step in pre-mRNA 3 end processing. Here, we characterize CF I m 68 subnuclear distribution and mobility. Fluorescence microscopy reveals that in addition to paraspeckles CF I m 68 accumulates in structures that partially overlap with nuclear speckles. Analysis of synchronized cells shows that CF I m 68 distribution in speckles and paraspeckles varies during the cell cycle. At an ultrastructural level, CF I m 68 is associated with perichromatin fibrils, the sites of active transcription, and concentrates in interchromatin granules-associated zones. We show that CFI m 68 colocalizes with bromouridine, RNA polymerase II, and the splicing factor SC35. On inhibition of transcription, endogenous CF I m 68 no longer associates with perichromatin fibrils, but it can still be detected in interchromatin granules-associated zones. These observations support the idea that not only splicing but also 3 end processing occurs cotranscriptionally. Finally, fluorescence recovery after photobleaching analysis reveals that the CF I m 68 fraction associated with paraspeckles moves at a rate similar to the more dispersed molecules in the nucleoplasm, demonstrating the dynamic nature of this compartment. These findings suggest that paraspeckles are a functional compartment involved in RNA metabolism in the cell nucleus. INTRODUCTIONMost functional mRNAs of eukaryotic genes are generated from their primary transcripts (pre-mRNAs) through RNA splicing and 3Ј end polyadenylation. Removal of introns occurs in the spliceosome, a complex composed of five small ribonucleoprotein particles (U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles [snRNPs]) and many non-snRNPs splicing factors, including members of the arginine-serine (SR) family of proteins. The mature 3Ј ends of mRNAs are generated by endonucleolytic cleavage of the pre-mRNA followed by polyadenylation of the upstream cleavage product. Biochemical studies have identified six factors required for efficient processing in vitro: the cleavage and polyadenylation specificity factor (CPSF), the cleavage stimulation factor (CstF), and two cleavage factors, mammalian cleavage factor I m [CF I m ] and CF II m , are necessary for the cleavage reaction. Polyadenylation requires in addition to CPSF, poly(A) polymerase, and the nuclear poly(A) binding protein 1 (PABPN1, previously called PAB II, for review, see Wahle and Rü egsegger, 1999). Other proteins involved in either transcription, such as the carboxy-terminal domain of RNA polymerase II, or capping (nuclear cap-binding complex) and splicing (U2AF65) have been shown to greatly enhance the efficiency of the first step of the reaction (Flaherty et al., 1997;Hirose and Manley, 1998;Millevoi et al., 2002).The mammalian cell nucleus is a highly structured and dynamic compartment. Many nuclear factors are localized in morphologically well-defined structural units that include the nucleolus and several "nuclear bodies," such as the Cajal ...

Mammalian pre-mRNA 3′ End Processing Factor CF Im68 Functions in mRNA Export

“…In this report we provide first evidence that the 68-kDa subunit of CF I m , a pre-mRNA 3Ј end processing factor, interacts with the mRNA export receptor NXF1 and stimulates mRNA export. On the basis of our observations and on previous data showing that CF I m 68 is associated with BrdU-labeled nascent transcripts (Cardinale et al, 2007), we propose that CF I m 68 is loaded onto the pre-mRNA during cleavage and polyadenylation of the 3Ј end of the transcript and remains bound to the mRNA all the way to the cytoplasm where it is removed by the translation machinery. CF I m 68 may thus act as a mark of correct 3Ј end maturation and contribute to efficient mRNA export via NXF1 recruitment.…”

“…Therefore, it is possible that CF I m 68 is leaving the nucleus by "piggy-backing" on RNA molecules being exported by other factors, either in association with the 25-kDa subunit or because of a direct interaction via the C-terminal domain. To determine whether shuttling of CF I m 68 depends on mRNA synthesis, we performed heterokaryon assays in the presence of actinomycin D. As shown in Figure 2B, GFP-CF I m 68 shuttling is blocked in presence of the transcription inhibitor, whereas relocalization of the protein in cap-like structures around the nucleoli of the human cell can be observed, as previously described (Cardinale et al, 2007; Figure 2B). Although we cannot formally rule out the possibility that actinomycin D could affect the synthesis of a short-lived protein required for export, the most likely conclusion of these experiments is that CF I m 68 shuttling is dependent on mRNA synthesis and possibly export.…”

“…The 68-kDa protein possesses an N-terminal RNPtype RNA recognition motif (RRM) and an RS-like C-terminal region enriched in RS/D/E dipeptides that is reminiscent of the RS domain of SR proteins. The RS-like domain of CF I m 68-kDa subunit is sufficient for the localization in nuclear speckles (and in the nucleoplasm) and mediates the interaction in vitro with a subset of shuttling SR proteins (Dettwiler et al, 2004;Cardinale et al, 2007).…”

Nuclear pre-mRNA 3′-end processing regulates synapse and axon development in C. elegans

“…An increasing number of studies have shown that dynamic and distinct nuclear compartments are likely to be associated with various pre-mRNA processing activities in many cell types (Boronenkov et al, 1998;Cardinale et al, 2007;Huang and Spector, 1991;Huang and Spector, 1992;Lamond and Spector, 2003;Mellman et al, 2008;Mintz and Spector, 2000;Politz et al, 2006). SYDN-1 could negatively regulate NpolyA activity on selected transcripts via direct interaction with PFS-2, or indirectly via additional proteins.…”

Arginine methylation in subunits of mammalian pre-mRNA cleavage factor I

“…The CF I m 25 dimer together with one of the two larger subunits is sufficient to stimulate cleavage, although it is not known in what function the large subunits differ (Rüegsegger et al 1996(Rüegsegger et al , 1998. Analysis of the subnuclear localization of CF I m 68 has identified this protein in speckles and paraspeckles and its location varies during the cell cycle (Cardinale et al 2007). Moreover, sequence-specific RNA binding of the CF I m 68 subunit to motifs within the 39 UTR of its own premRNA has been reported to suppress cleavage (Brown and Gilmartin 2003).…”