Both phosphorylation and dephosphorylation of ASF/SF2 are required for pre-mRNA splicing in vitro

Wenhong Cao, Sharon F. Jamison, Mariano Garcia-Blanco

Research output: Contribution to journalArticle

136 Citations (Scopus)

Abstract

The splicing reaction that removes introns from pre-messenger RNAs requires the assembly of the spliceosome on the nascent transcript, proper folding of the substrate-enzyme complex, and finally, two transesterification reactions. These stages in the splicing reaction must require careful orchestration. Here we show data that suggest that the sequential phosphorylation and dephosphorylation of SR proteins mark the transition between stages in the splicing reaction. Many data had already led to the idea that phosphorylation of SR proteins could modulate their activity, when we showed that dephosphorylation of these proteins abrogates their activity in a reaction measuring conversion of pre-spliceosomes to spliceosomes (Roscigno RF, Garcia-Blanco MA, 1995, RNA 1:692-706). Subsequently, Xiao and Manley (1997, Genes and Dev 11:334-344) showed that phosphorylated ASF/SF2, but not mock-phosphorylated ASF/SF2, activates the splicing of HIV tat pre- mRNA in reactions challenged with excess random RNA. Here we confirm and extend these two findings. Phosphorylated ASF/SF2 efficiently complemented an SR protein-deficient HeLa S100 extract in promoting the splicing of an adenovirus-2-derived pre-messenger RNA, whereas unphosphorylated ASF/SF2 did not. Moreover, we show that, whereas unphosphorylated ASF/SF2 inhibited splicing in HeLa nuclear extracts, phosphorylation of the ASF/SF2 reversed the inhibition and enhanced splicing. We also present data that shows that dephosphorylation of ASF/SF2 is required for the first transesterification reaction once the spliceosome has assembled. Thiophosphorylated ASF/SF2, which cannot be readily dephosphorylated, can promote spliceosome assembly, but cannot promote the first transesterification reaction. These data, together with other observations, indicate for the first time a requirement for SR protein dephosphorylation in pre-messenger RNA splicing in vitro.

Original languageEnglish (US)
Pages (from-to)1456-1467
Number of pages12
JournalRNA
Volume3
Issue number12
StatePublished - Dec 1997
Externally publishedYes

Fingerprint

Spliceosomes
RNA Precursors
Phosphorylation
Proteins
RNA
Conversion Disorder
RNA Splicing
Messenger RNA
Adenoviridae
Introns
In Vitro Techniques
HIV
Enzymes
Genes

Keywords

  • MRNA splicing mechanism
  • MRNA splicing signals
  • Phosphorylation
  • SR domain

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology

Cite this

Both phosphorylation and dephosphorylation of ASF/SF2 are required for pre-mRNA splicing in vitro. / Cao, Wenhong; Jamison, Sharon F.; Garcia-Blanco, Mariano.

In: RNA, Vol. 3, No. 12, 12.1997, p. 1456-1467.

Research output: Contribution to journalArticle

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