The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells

GFP-tagged snRNP components reveal the dynamics and rate for spliceosome assembly in vivo

Formování a recyklace funkčních sestřihových komplexů in vivo

Institute of Cell Biology and Pathology First Faculty of Medicine Charles University in PragueÚstav buněčné biologie a patologie 1. LF UK v PrazeFirst Faculty of Medicine1. lékařská fakult

Production of antibodies to a recombinant potato mop-top virus protein TGBp1

Katedra biochemieDepartment of BiochemistryPřírodovědecká fakultaFaculty of Scienc

Spliceosomal Small Nuclear Ribonucleoprotein Particles Repeatedly Cycle through Cajal Bodies

The Cajal body (CB) is a nuclear structure closely associated with import and biogenesis of small nuclear ribonucleoprotein particles (snRNPs). Here, we tested whether CBs also contain mature snRNPs and whether CB integrity depends on the ongoing snRNP splicing cycle. Sm proteins tagged with photoactivatable and color-maturing variants of fluorescent proteins were used to monitor snRNP behavior in living cells over time; mature snRNPs accumulated in CBs, traveled from one CB to another, and they were not preferentially replaced by newly imported snRNPs. To test whether CB integrity depends on the snRNP splicing cycle, two human orthologues of yeast proteins involved in distinct steps in spliceosome disassembly after splicing, hPrp22 and hNtr1, were depleted by small interfering RNA treatment. Surprisingly, depletion of either protein led to the accumulation of U4/U6 snRNPs in CBs, suggesting that reassembly of the U4/U6·U5 tri-snRNP was delayed. Accordingly, a relative decrease in U5 snRNPs compared with U4/U6 snRNPs was observed in CBs, as well as in nuclear extracts of treated cells. Together, the data show that particular phases of the spliceosome cycle are compartmentalized in living cells, with reassembly of the tri-snRNP occurring in CBs