p54(nrb) associates with the 5' splice site within large transcription/splicing complexes - PubMed (original) (raw)

p54(nrb) associates with the 5' splice site within large transcription/splicing complexes

Sei Kameoka et al. EMBO J. 2004.

Abstract

The functional coupling of transcription and splicing has been reported both in vivo and in vitro, but the molecular mechanisms governing these interactions remain largely unknown. Here we show that p54(nrb), a transcription/splicing factor, associates with the 5' splice site (SS) within large complexes present in HeLa cell nuclear extracts, in which the hyperphosphorylated form of RNA polymerase II (RNAPIIO) is associated with U1 or U1 and U2 snRNPs. These RNAPIIO-snRNP complexes also contain other transcription/splicing factors, such as PSF and TLS, as well as transcription factors that interact with RNAPIIO during elongation, including P-TEFb, TAT-SF1 and TFIIF. The presence of these factors in functional elongation complexes, demonstrated using an immobilized DNA template assay, strongly suggests that the RNAPIIO-snRNP complexes reflect physiologically relevant interactions between the transcription and splicing machineries. Our finding that both p54(nrb) and PSF, which bind the C-terminal domain of the largest subunit of RNAPII, can interact directly with the 5' SS indicates that these factors may mediate contacts between RNAPII and snRNPs during the coupled transcription/splicing process.

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Figures

Figure 1

Figure 1

Crosslinking profile of the BP-derivatized 5′SS RNA. (A) Structure of the 32P-labeled, BP-derivatized 14-nt 5′SS RNA. (B) The 5′SS RNA was incubated with HeLa NE in the absence (lanes 1, 3) or presence (lanes 2, 4) of ATP and in the presence (lanes 1, 2) or absence (lanes 3, 4) of U1-blocking DNA. (C) Wild-type 5′SS RNA (WT) or 5′SS RNAs with single point mutations at intron pos. +1 or +2 was incubated with HeLa NE in the absence of ATP or U1-blocking DNA. (D) The 5′SS crosslinks were immunoprecipitated under denaturing conditions with α-Sm (Y12) antibodies. Crosslinked products were resolved in 11% SDS gels. Positions of the molecular weight markers and the 5′SS crosslinks are indicated.

Figure 2

Figure 2

The 5′SS:p54/56 crosslink corresponds to p54nrb. (A) The 5′SS:p54/56 crosslinks were treated with RNase A (lane 2) or RNase A and phosphatase (CIP) (lane 3) and resolved in an 8% SDS gel. (B) The 5′SS crosslinks were immunoprecipitated under denaturing (lanes 1–4) or native (lanes 5 and 6) conditions using the indicated antibodies and resolved in an 11% SDS gel. (C) The 5′SS:p54nrb and 5′SS:hPrp28 crosslinks were purified from an SDS gel, mixed (lane 1), immunoprecipitated with pre-immune serum (lane 2) or the indicated antibodies (lanes 3 and 4) and resolved in an 8% SDS gel. (D) Diagram of the human PSF and p54nrb sequences. The two RRMs, the proline/glutamine-rich regions (PQ), the 293-aa internal region of high similarity between p54nrb and PSF, the sequences of three Lys-C peptides and the mapped region of the 5′SS crosslink within p54nrb (aa 18–53) are indicated.

Figure 3

Figure 3

The 5′SS:p54nrb crosslink exists exclusively in large complexes containing U1 snRNP. (A) Standard binding reactions in the presence (lanes 1–4) or absence (lanes 5–7) of the 5′SS RNA, in the absence (lanes 1,3) or presence (lanes 2,4–7) of ATP and in the presence (lanes 1,2) or absence (lanes 3–7) of U1-blocking DNA were resolved in a native 4% gel. snRNP complexes were detected by autoradiography (lanes 1–4) or Northern hybridization with U1, U2 or U4 probes (lanes 5–7). Positions of complexes are indicated. (B) The 5′SS crosslinks were resolved in a native 4% gel in the first dimension and in an 11% SDS gel in the second. Positions of the free 5′SS and the crosslinks are indicated.

Figure 4

Figure 4

X1 and X2 complexes can be separated in a glycerol gradient. A 150-μl crosslinking reaction performed in the presence of ATP and absence of U1-blocking DNA was separated in a 10–30% glycerol gradient. In all, 10 μl of each fraction was resolved in either a 4% native gel (A) or an 11% SDS gel (B). (C) Fractions 4 and 7 of the glycerol gradient were immunoprecipitated under native conditions using the indicated antibodies, and the bound material was resolved in an 11% SDS gel. The identity of the ∼70 kDa signal generated with the α-TLS antibody (lane 21) has not been determined. Positions of the 5′SS crosslinks are indicated.

Figure 5

Figure 5

The p54nrb crosslink in X1 and X2 complexes is associated with RNAPIIO. A crosslinking reaction performed in the presence of ATP and in the absence (A) or presence (B) of U1-blocking DNA was immunoprecipitated under native conditions using the indicated α-RNAPII antibodies and the bound material resolved in an 11% SDS gel. The identity of the ∼65 kDa signal (e.g. lane 3 in panel A) has not been determined. (C) HeLa NE was incubated with ATP in the absence of the 5′SS RNA and immunoprecipitated as in (A). The bound RNA was analyzed by Northern hybridization with a mixture of U1, U2, U4, U5 and U6 snRNA probes. Positions of snRNAs are indicated. Although co-migrating with U6, the signal indicated by an arrow in lane 4 represents a degradation product of U1 snRNA.

Figure 6

Figure 6

X1 and X2 complexes contain several transcription elongation factors. A crosslinking reaction performed in the presence of ATP was separated in a glycerol gradient, and fractions containing X1 (fraction 4) and X2 (fraction 7) complexes were immunoprecipitated under native conditions using the indicated antibodies and the bound material was resolved in an 11% SDS gel.

Figure 7

Figure 7

X1 and X2 complexes contain a subset of factors present in functional transcription ECs. (A) Schematic representation of the immobilized Ad20 and Ad22 DNA templates, showing the AdMLP, the two leading exons of the adenovirus major late transcription unit (black boxes), the start site for RNAPII transcription and the unique _Pvu_II restriction sites. (B) Western blot analysis of protein factors bound to template fragments released from beads lacking DNA or linked to the Ad20 or Ad22 templates using the indicated antibodies. (C) Northern blot analysis of template fragments released from beads lacking DNA or linked to the Ad20 or Ad22 templates using probes for all the five U snRNAs and the Ad22 transcript.

Figure 8

Figure 8

Schematic composition of X1 and X2 complexes. Relative positions of the indicated components are arbitrary.

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