Reconstitution of the U1 small nuclear ribonucleoprotein particle.

摘要: Abstract Although the U1 small nuclear ribonucleoprotein particle (snRNP) was first mRNA-splicing cofactor to be identified, manner in which it functions splicing is not precisely understood. Among information required understand how snRNP participates splicing, will necessary know its structure. Here we describe vitro reconstitution of a that possesses properties native snRNP. 32P-labeled RNA transcribed from an SP6 promoter-human gene clone and incubated HeLa S100 fraction. A formed displayed same sedimentation coefficient (approximately 10S) buoyant density (1.40 g/cm3) as The latter value reflects ability withstand isopycnic banding Cs2SO4 without prior fixation, property shared by reconstituted reacted with both Sm RNP monoclonal antibodies, showing these two classes proteins were present. Moreover, found display characteristic Mg2+ switch nuclease sensitivity previously described for snRNP: open, nuclease-sensitive conformation at low concentration (3 mM) more compact, nuclease-resistant organization higher (15 mM). majority did contain hypermethylated caps, pseudouridine, or ribose 2-O-methylation, enigmatic posttranscriptional modifications are essential particle. extreme 3' end (18 nucleotides) reconstitution, but loop II (nucleotides 64 77) not. Interestingly, 5' recognizes pre-mRNA splice sites reconstruction.