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

Abstract

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

CRE promoter sites modulate alternative splicing via p300-mediated histone acetylation

“…[6][7][8] In vivo splicing is achieved within tens of seconds suggesting most introns are spliced out while RNA is still attached to Pol ll and the DNA template. [9][10][11] Consistent with this data, splicing factors have been shown to be recruited to the site of pre-mRNA synthesis and bind the nascent pre-mRNA [12][13][14] and 80% of active spliceosomes are attached to chromatin. 15 This coupling of premRNA splicing and Pol II transcription brings another layer of regulation.…”

The timing of pre-mRNA splicing visualized in real-time

“…24 FRAP analysis further revealed that core spliceosomal snRNP proteins have a residence time of 15-30 s in the nucleoplasm, where spliceosomal snRNPs are thought to interact predominantly with pre-mRNA. 25 Based on these results it was suggested that splicing can be accomplished within 30 s, which is significantly more rapid than previously reported. 25 Following the advent of genetically encoded fluorescent protein tags, Belmont and colleagues pioneered a method to image chromatin dynamics in vivo.…”

“…25 Based on these results it was suggested that splicing can be accomplished within 30 s, which is significantly more rapid than previously reported. 25 Following the advent of genetically encoded fluorescent protein tags, Belmont and colleagues pioneered a method to image chromatin dynamics in vivo. They introduced bacterial lac operator repeats into the genome of yeast and mammalian cells that expressed a GFP-lac repressor fusion protein.…”

The nuclear cap-binding complex interacts with the U4/U6·U5 tri-snRNP and promotes spliceosome assembly in mammalian cells

“…We addressed total cellular pre-mRNA by monitoring snRNP dynamics in the nucleoplasm; complementary to this, we used an integrated, inducible transcription unit (E3) containing three exons and two introns of the β-globin gene ( Fig. 4A; Huranova et al 2010;Pabis et al 2010;Brody et al 2011). E3 mRNA contains the MS2 binding site in the 3 ′ UTR, such that active transcription sites can be monitored in living cells with a fluorescently tagged MS2-binding protein.…”