Separation of splicing factor SF3 into two components and purification of SF3a activity

Brosi, Reto; Hauri, Hans Peter; Krämer, Angela

doi:10.1016/s0021-9258(19)85380-2

Cited by 116 publications

(43 citation statements)

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“…In vitro, the 17S U2 snRNP is reconstituted by incubation of the 12S U2 snRNP with splicing factors (SF) 3a and 3b (Brosi et al, 1993a) that were initially isolated as non-snRNP proteins (Brosi et al, 1993b). The 12S U2 snRNP and SF3b associate to form a particle of 15S; addition of SF3a to the 15S U2 snRNP results in the assembly of the 17S particle.…”

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confidence: 99%

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Combined Biochemical and Electron Microscopic Analyses Reveal the Architecture of the Mammalian U2 snRNP

Krämer

Grüter

Gröning

et al. 1999

The Journal of Cell Biology

114

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The 17S U2 small nuclear ribonucleoprotein particle (snRNP) represents the active form of U2 snRNP that binds to the pre-mRNA during spliceosome assembly. This particle forms by sequential interactions of splicing factors SF3b and SF3a with the 12S U2 snRNP. We have purified SF3b and the 15S U2 snRNP, an intermediate in the assembly pathway, from HeLa cell nuclear extracts and show that SF3b consists of four subunits of 49, 130, 145, and 155 kD. Biochemical analysis indicates that both SF3b and the 12S U2 snRNP are required for the incorporation of SF3a into the 17S U2 snRNP. Nuclease protection studies demonstrate interactions of SF3b with the 5′ half of U2 small nuclear RNA, whereas SF3a associates with the 3′ portion of the U2 snRNP and possibly also interacts with SF3b. Electron microscopy of the 15S U2 snRNP shows that it consists of two domains in which the characteristic features of isolated SF3b and the 12S U2 snRNP are conserved. Comparison to the two-domain structure of the 17S U2 snRNP corroborates the biochemical results in that binding of SF3a contributes to an increase in size of the 12S U2 domain and possibly induces a structural change in the SF3b domain.

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confidence: 99%

“…4 B) and the intron branch site. This reaction requires the 12S U2 snRNP plus SF3a and SF3b (Brosi et al, 1993b) or the isolated 17S U2 sn-RNP (Behrens et al, 1993a); the 12S U2 snRNP alone is inactive in complex assembly. Thus, SF3a and SF3b are essential for prespliceosome assembly in combination with the 12S U2 snRNP.…”

mentioning

confidence: 99%

Combined Biochemical and Electron Microscopic Analyses Reveal the Architecture of the Mammalian U2 snRNP

Krämer

Grüter

Gröning

et al. 1999

The Journal of Cell Biology

114

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“…The U2 small nuclear ribonucleoprotein particle (snRNP) is a vital component of the human spliceosome, which is the macromolecular complex responsible for pre-mRNA splicing during eukaryotic gene expression. U2 snRNP is composed of the U2 snRNA, core proteins, and SF3A and SF3B subcomplexes [ 1 , 2 ]. During splicing, U2 snRNP is responsible for branch point recognition via base-pairing interaction with the intron to select the branch point adenosine that participates in the first chemical step of splicing [ 3 , 4 ].…”

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confidence: 99%

U2 snRNA structure is influenced by SF3A and SF3B proteins but not by SF3B inhibitors

et al. 2021

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U2 snRNP is an essential component of the spliceosome. It is responsible for branch point recognition in the spliceosome A-complex via base-pairing of U2 snRNA with an intron to form the branch helix. Small molecule inhibitors target the SF3B component of the U2 snRNP and interfere with A-complex formation during spliceosome assembly. We previously found that the first SF3B inhibited-complex is less stable than A-complex and hypothesized that SF3B inhibitors interfere with U2 snRNA secondary structure changes required to form the branch helix. Using RNA chemical modifiers, we probed U2 snRNA structure in A-complex and SF3B inhibited splicing complexes. The reactivity pattern for U2 snRNA in the SF3B inhibited-complex is indistinguishable from that of A-complex, suggesting that they have the same secondary structure conformation, including the branch helix. This observation suggests SF3B inhibited-complex instability does not stem from an alternate RNA conformation and instead points to the inhibitors interfering with protein component interactions that normally stabilize U2 snRNP’s association with an intron. In addition, we probed U2 snRNA in the free U2 snRNP in the presence of SF3B inhibitor and again saw no differences. However, increased protection of nucleotides upstream of Stem I in the absence of SF3A and SF3B proteins suggests a change of secondary structure at the very 5′ end of U2 snRNA. Chemical probing of synthetic U2 snRNA in the absence of proteins results in similar protections and predicts a previously uncharacterized extension of Stem I. Because this stem must be disrupted for SF3A and SF3B proteins to stably join the snRNP, the structure has the potential to influence snRNP assembly and recycling after spliceosome disassembly.

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“…79: Απλοποιηµένο σχήµα της βιογένεσης των U snRNPs snRNP (Will CL et al, 1999). Ο SF3b είναι ένα ιδιαίτερα σταθερό σύµπλοκο (Brosi R et al, 1993), απαραίτητο για το σχηµατισµό του pre-spliceosome (Brosi R et al, 1993) και παραµένει στο spliceosome µέχρι και την ολοκλήρωση της διαδικασίας της συναρµογής (Champion-Arnaud P & Reed R, 1994). Εκτός της SAP145, ο SF3b περιλαµβάνει τουλάχιστον 7 επιπλέον πρωτεΐνες έχοντας συνολικό µοριακό βάρος 450kD (Will CL et al 2001 (Yagi H et al, 2003).…”

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“…2000) ενώ οι κατ' αναλογία εκτιµήσεις βάσει του αντίστοιχου αριθµού των γονιδίων των χρωµοσωµάτων 20, 21 και 22 ήταν 31,500(Deloukas P et al, 2001),44.000(Hattori Μ et al, 2000) και 45.000(Dunham I et al, 1999), αντίστοιχα. Ακόµα και η εκτίµηση βάσει της πρώτης έκδοσης της αλληλουχίας του γονιδιώµατος είχε µεγάλη απόκλιση από τη σηµερινή.…”

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Φυσικός χάρτης προς αλληλούχηση της περιοχής q23.3 - q25.3 του χρωμοσώματος 10 του ανθρώπου και ανάλυση του γονιδίου FRA10AC1

Sarafidou¹,

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Φυσικός χάρτης προς αλληλούχηση της περιοχής q23.3-q25.3 του χρωµοσώµατος 10 του ανθρώπου και ανάλυση του γονιδίου FRA10AC1» ΘΕΟΛΟΓΙΑ ΣΑΡΑΦΙ∆ΟΥ Εργαστήριο Μοριακής Γενετικής Ανθρώπου Επιβλέπων Καθηγητής: Ν. Κ. Μοσχονάς Ηράκλειο, Νοέµβριος 2005 UNIVERSITY OF CRETE SCHOOL OF SCIENCE AND ENGINEERING DEPARTMENT OF BIOLOGY AND INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY Ph. D Thesis «Sequence-ready physical map of human chromosomal region 10q23.3-q25.1 and analysis of FRA10AC1 gene »

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Separation of splicing factor SF3 into two components and purification of SF3a activity

Cited by 116 publications

References 0 publications

Combined Biochemical and Electron Microscopic Analyses Reveal the Architecture of the Mammalian U2 snRNP

Combined Biochemical and Electron Microscopic Analyses Reveal the Architecture of the Mammalian U2 snRNP

U2 snRNA structure is influenced by SF3A and SF3B proteins but not by SF3B inhibitors

Φυσικός χάρτης προς αλληλούχηση της περιοχής q23.3 - q25.3 του χρωμοσώματος 10 του ανθρώπου και ανάλυση του γονιδίου FRA10AC1

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