©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Mammalian Activity Required for the Second Step of Pre-messenger RNA Splicing (*)

Laura A. Lindsey (1)(§), Allen J. Crow (1) (3)(¶), Mariano A. Garcia-Blanco (1) (3) (2)(**)

From the (1) Departments of Molecular Cancer Biology, (2) Microbiology, and (3) Medicine, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Splicing of precursors to messenger RNAs occurs via a two-step mechanism. In the first step, the 5`-exon is released concomitant with the production of a lariat intermediate, and in the second step, the exons are joined, releasing the intron in the form of a lariat product. Several gene products of the yeast Saccharomyces cerevisiae have been shown to be required exclusively for the second step. Although mammalian proteins have been implicated in the second step of splicing, none have been shown to act only at this step. We identify here the first mammalian activity shown to be exclusively required for the second step. The activity was shown to increase by 5-fold the rate for this splicing step, whereas it had no effect on the rate of the first step. The activity was not affected by treatment with micrococcal nuclease, whereas it is sensitive to heating to 55 °C, suggesting that it is not dependent on an RNA, but more likely is a protein. The second step activity was separated from other factors required for the first step and from PSF, a splicing factor thought to have a second step activity. The activity does not require ATP hydrolysis, suggesting that it acts at a late stage of the second step of splicing.


INTRODUCTION

Introns are removed from precursors to messenger RNAs (pre-mRNAs) in a precise process termed pre-mRNA splicing (1) . The mechanism for this removal has been conserved throughout evolution of eukaryotes and is related to both group I and II self-splicing mechanisms (2, 3) . Both pre-mRNA and group II splicing proceed via a two-step mechanism that involves the formation of a 2`,5`-phosphodiester bond. In the first step (also step I) of pre-mRNA splicing, cleavage at the 5`-splice site yields a ``free'' 5`-exon and a lariat intermediate (reviewed in Refs. 1 and 2). The lariat intermediate has the 5`-position of the first nucleotide of the intron, usually a G, involved in a 2`,5`-phosphodiester link with the branch nucleotide in the intron. In the second step (also step II), the 5`-exon is ligated to the 3`-exon with displacement of the intron as a lariat product. Both these steps are transesterification reactions and involve no transfer of phosphates from ATP (4) . Recent studies have shown that the reaction mechanisms of both steps proceed via S2 displacements (5) . This mechanism suggests that the two steps of splicing probably occur in two distinct catalytic sites in the spliceosome (5) , which is consistent with the existence of factors acting exclusively at one of the two sites.

Several gene products of the yeast Saccharomyces cerevisiae have been shown to be required only for the second step of splicing (reviewed in Refs. 1 and 2). PRP16, a 120-kDa protein with a DEAH box motif (6) , is required for step II (7, 8, 9, 10) . PRP16 has RNA-dependent NTPase and dNTPase activities, and its effect on step II requires NTP binding and possibly hydrolysis (11) . The broad specificity range for these two activities argues that PRP16 may be the last NTPase required in the splicing reaction. PRP16 associates with the spliceosome after the completion of the first step of splicing and mediates a conformational change in the spliceosome (11) .

Another well studied factor required for the second step is PRP18 (12-15). This 28-kDa protein without identifiable motifs is associated with U5 snRNP()(13) . Extracts depleted of PRP18 show an 80-fold slower rate for step II, whereas the rate of step I is not significantly altered relative to wild-type extract (14) . The splicing intermediates formed in the depleted extracts can be chased to mRNA and lariat product by the addition of recombinant PRP18 in the absence of detectable levels of ATP (14) . Also required for step II are PRP17 (SLU4) and SLU7 (16, 17) . Synthetic lethality experiments suggest the existence of functional interactions between PRP16, PRP17, PRP18, SLU7, and U5 snRNA (17) .

Krainer and Maniatis (18) described two activities required for the second step of splicing: SF3 and SF4A. SF3 was described as a heat-sensitive activity resistant to micrococcal nuclease treatment, and SF4A was not characterized. More recently 15 proteins have been shown to be associated with purified spliceosomal complex C1, in which splicing is arrested after step I (19) . It is reasonable to assume that some of these will be required for the second step of splicing. PSF, a protein previously described as a splicing factor required for pre-spliceosome formation (20) , was shown to be one of these complex C1-associated proteins (19) . Data both in the original publication by Patton et al.(20) and in Gozani et al.(19) suggest a possible involvement of PSF in the second step of splicing. No mammalian activity has been subjected to rigorous analysis in order to show a second step effect independent of earlier effects such as has been accomplished for the yeast factors PRP16 and PRP18.

In this report, we present the results of the study of a mammalian activity required for the efficient catalysis of the second step of splicing. Independent measurement of the rates of the two steps of splicing was accomplished by determining initial rates of these reactions. These measurements argue that this mammalian activity is required exclusively for step II. Characterization of the activity is described, and its relationship to other mammalian and yeast factors is discussed. We show that the activity does not require ATP and is not inhibited by the addition of an excess of nonhydrolyzable ATP analogues. Finally, we show that spliceosomes formed in the absence of this activity can be chased through step II.


EXPERIMENTAL PROCEDURES

Plasmids, RNAs, and Nuclear Extracts

pPIP7.A has been described previously (21) . pPIP7.A contains a 55-nucleotide 5`-exon, a 125-nucleotide intron, and a 56-nucleotide 3`-exon derived from the adenovirus major late promoter. PIP7.A RNA was synthesized as described previously (22, 23) . Briefly, pPIP7.A was linearized by digestion with HindIII. The linearized template was transcribed with T7 RNA polymerase (New England Biolabs Inc.) in the presence of [-P]UTP at a final specific activity of 300 Ci/mmol. Nuclear extract was prepared from HeLa cells by the method of Dignam et al.(24) .

Nuclear Extract Fractionation

34 ml of HeLa nuclear extract (in Buffer D (20 mM Hepes (pH 7.9), 20% (v/v) glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol) with 100 mM KCl) containing 537 mg of protein were loaded onto a DEAE-Sephacel column (cross-sectional area = 4.96 cm; height = 3.36 cm; volume = 16.5 ml) at a rate of 2.5 ml/h/cm. The DEAE-Sephacel column had been equilibrated in Buffer D with 100 mM KCl. A total of 25 5-ml fractions were collected. Fractions 4-8, containing 40% of the protein, were found to have second step activity and were then pooled and designated DEAE I. The DEAE-Sephacel column was then eluted with Buffer D with 500 mM KCl at 2.5 ml/h/cm, and 5-ml fractions were collected. A 10-µl aliquot of each of the fractions eluted from the column with Buffer D with 500 mM KCl was analyzed for protein content, and fractions 2-15 were found to contain the vast majority of protein (60% of protein loaded). These fractions were pooled and dialyzed into Buffer D with 100 mM KCl and termed DEAE II. 24 ml of the DEAE I fraction containing 211 mg of protein were loaded onto a CM-Sepharose CL-6B column (cross-sectional area = 1.77 cm; height = 6.2 cm; volume = 11 ml) at a rate of 5 ml/h/cm. The column was washed with 15 ml of Buffer D with 100 mM KCl and then step-eluted with 15 ml each of Buffer D with 250 mM KCl, Buffer D with 500 mM KCl, and Buffer D with 1 M KCl. 5-ml fractions were collected and analyzed for second step activity. Fractions 2-6, containing 75% of the protein, were pooled and termed CM-Sepharose flow-through (CMFT). 2 ml of CMFT (12 mg) were loaded onto a Sephacryl S-300 HR column (cross-sectional area = 2.0 cm; height = 35 cm; volume = 70 ml) at a rate of 2 ml/h/cm. The Sephacryl column had been standardized and then equilibrated in Buffer D with 100 mM KCl. A total of 70 1-ml fractions were collected and assayed for step II activity. Fractions 26-29 had activity. The overall -fold purification can only be estimated roughly to be on the order of 10-fold. The activity is remarkably stable; little decay was observed in crude fractions stored at -70 °C for over 1 year.

Splicing Reactions

Splicing reactions were run as described previously (25) . Splicing reactions were run at 30 °C for the indicated length of time with final concentrations of 64 mM KCl, 2 mM MgCl, 1 mM ATP, 5 mM creatine phosphate, and 12.8% glycerol (v/v). In vitro splicing products and intermediates were detected by extracting the RNA and analyzing it on denaturing 15% polyacrylamide gels (25) . The splicing products and intermediates were visualized by autoradiography using Hyperfilm (Amersham Corp.). For splicing complex formation, reactions were incubated at 30 °C for the indicated length of time, placed on ice, and supplemented with heparin (Sigma H-3125) to a final concentration of 0.5 mg/ml. Splicing complexes were analyzed by electrophoresis on nondenaturing 4% polyacrylamide gels buffered with 50 mM Tris/glycine and run at 200 V (12.5 V/cm) at room temperature for 3 h (26). The gels were dried under vacuum at 80 °C for 1 h, and splicing complexes were visualized by autoradiography using Hyperfilm.

Fractions were assayed for the ability to catalyze the second step of splicing in two ways, a simultaneous assay and a sequential assay. In the simultaneous assay, DEAE II and the fraction being analyzed for second step activity were combined prior to the splicing reaction. In the sequential assay, DEAE II was added, and the splicing reaction was run for the indicated length of time. At that point, the fraction to be analyzed for second step activity was added to the original splicing reaction. The mixture was then incubated at 30 °C for the indicated length of time. In vitro splicing products and intermediates were analyzed as described above.

Microccocal Nuclease Digestion

The CMFT fraction was digested with micrococcal nuclease in the following manner. A 50-µl aliquot of CMFT was incubated at 30 °C for 30 min with 0.5 µl of 4 units/µl micrococcal nuclease in the presence of 1 mM CaCl. To stop the digestion, 6 µl of 100 mM EGTA were added, and the reaction mixture was incubated for an additional 2 min at 30 °C. A second 50-µl aliquot of CMFT was mock-treated by supplementing it with 1 mM CaCl and incubating at 30 °C for 30 min. Aliquots of nuclear extract were treated in the same manner.

Depletion of ATP

To deplete the reactions of ATP after initial incubation with DEAE II, hexokinase (Sigma) and glucose were added to final concentrations of 25 µg/ml and 12 mM, respectively (27) . This was allowed to incubate for 10 min at 30 °C. The reaction was chased with DEAE I for 90 min at 30 °C. To follow ATP levels, the reactions were supplemented with [-P]ATP (12 µCi/ml). 1-µl aliquots were taken for each condition before and after incubations and incubated with 30 µl of 15% (w/v) chilled trichloroacetic acid for 15 min on ice. After spinning at 16,000 g for 15 min, the supernatant fraction was collected and neutralized with an equal volume of 1 N NaOH. 0.5 µl was spotted on polyethyleneimine-cellulose thin-layer chromatography plates; the plates were developed with 1 M LiCl; and products were visualized by autoradiography (28) .

Gradient Purification of Spliceosomes and Chase

Procedures were the same as described previously (22) with the following exceptions. The 100-µl splicing reaction contained DEAE II rather than nuclear extract and was incubated for 70 min at 30 °C. This was loaded onto a 1.9-ml 15-30% (w/v) glycerol gradient, and 10-200-µl fractions were collected.

Preparation of PSF-depleted Nuclear Extract and Western Blotting

PSF depletion was essentially as described previously (20). Nuclear extract (8.5 ml) was adjusted to 500 mM KCl in Buffer D and applied to a 4.5-ml poly(U)-agarose column and 1-ml fractions were collected. Flow-through material and 500 mM KCl were collected (fraction I). Step fractions were eluted with 1.0 M KCl (fraction II) and 1.0 M KCl and 2 M guanidinium Cl (fraction III), and the resultant peaks were dialyzed in Buffer D with 100 mM KCl. For Western blotting, protein was run on a 12.5% SDS-polyacrylamide gel and then transferred onto Immobilon affinity membranes (Millipore Corp.) in 20% methanol, 192 mM glycine, and 25 mM Tris. Following transfer, blots were blocked in 5% nonfat dry milk with phosphate-buffered saline and 0.1% Tween 20. The PSF antibody (kindly provided by Dr. J. Patton, Vanderbilt University) was diluted 1:5000 in the same buffer and incubated at room temperature for 1 h, followed by incubation with a peroxidase-conjugated secondary antibody (Amersham Corp.) for 1 h at room temperature. Visualization was with the ECL reagents from Amersham Corp. following their protocol.


RESULTS

A Mammalian Activity Required for the Second Step of Splicing

HeLa nuclear extracts were fractionated over DEAE-Sephacel to separate factors required for pre-mRNA splicing. Three fractions were collected: a flow-through/wash 100 mM KCl fraction (DEAE I), a fraction eluted with 500 mM KCl (DEAE II), and a fraction eluted with 1 M KCl (DEAE III). The DEAE I and II fractions were capable of reconstituting splicing with high efficiency (Fig. 1A, lane5) relative to the nuclear extract loaded onto the column (lanes1 and 2; note that lane1 is compressed at the origin). The DEAE I fraction had no detectable splicing activity (lane3). DEAE II was as active as the extract in catalyzing step I: the formation of a lariat intermediate and a free 5`-exon (lane4). The conversion of these intermediates to mature mRNA and lariat product of step II, however, was very low compared with extract and with the reactions containing both DEAE I and II fractions (lanes2 and 5). Therefore, DEAE II contained all the factors required to complete the first step of splicing, but was missing at least one activity required for the second step. DEAE I was capable of complementing DEAE II for this missing activity. It was reasonable to assume from this that the activity was not required for the first step of splicing.


Figure 1: An activity in the DEAE I fraction is required for the second step of splicing. A, in vitro splicing using the simultaneous assay described under ``Experimental Procedures.'' Splicing reactions were incubated in the absence of ATP with nuclear extract (NE; lane1) or in the presence of 1 mM ATP and 5 mM creatine phosphate with nuclear extract (lane2), DEAE I (lane3), DEAE II (lane4), or DEAE I + II (lane5). Note that lane1 is compressed near the origin and that the top part of lane2 deviates to the left. B, in vitro splicing using the sequential assay described under ``Experimental Procedures.'' Splicing reactions were incubated in the absence of ATP with nuclear extract (lane1) or in the presence of 1 mM ATP and 5 mM creatine phosphate with nuclear extract (lane2), DEAE II (lane3), DEAE II followed by chasing with DEAE I (lanes 4-6), or DEAE II followed by a mock chase with Buffer D (lanes 7-9). Splicing intermediates and products were purified and visualized as described under ``Experimental Procedures.'' From top to bottom, icons indicate the lariat intermediate, lariat product, pre-mRNA, spliced product, and free 5`-exon.



A second assay was used to detect this activity in DEAE I. Uniformly labeled pre-mRNA was incubated with DEAE II under splicing conditions for 45 min. The results were similar to those obtained before, and splicing was arrested after completion of step I (Fig. 1B, lane3). The reactions were divided into two, and one half was incubated further with Buffer D for 30, 60, or 90 min (lanes 7-9). This resulted in little accumulation of products. The other half was incubated with DEAE I for 30, 60, or 90 min, and the result was a dramatic conversion of intermediates into lariat products and mRNA (lanes 4-6). This result was consistent with the data shown in Fig. 1A, and it supported the idea that DEAE I had an activity involved exclusively in the second step of splicing. Moreover, the data suggest that the activity can interact with already assembled spliceosomes.

The DEAE Activity Affects the Rate of the Second Step

Experiments were performed to measure the initial rates of both steps in the splicing reactions to confirm that the activity in DEAE I was affecting the second step of splicing. DEAE I had absolutely no activity on its own, and therefore, the rate of both steps was zero (data not shown). The rate of the first step of splicing was measured for splicing reactions containing DEAE II versus reactions containing DEAE II + I. The assays were carried out as described for Fig. 1A (see ``Experimental Procedures''). The relative rates of accumulation of lariat intermediate and lariat product for the reactions were derived from data shown in Fig. 2 (A and B) and are plotted in Fig. 2C. Quantification was based on scanning on a PhosphorImager (Molecular Dynamics, Inc.). The level of the lariat intermediate or lariat product was normalized to the total RNA level to ensure that losses would not alter the results. The normalization did not have much of an effect on the rate measurements given that the different reactions had very similar levels of RNA. Measurements of free 5`-exon and mRNA gave comparable results, but these were deemed less reliable for quantification given the fact that in some cases the mRNA appeared as multiple bands (data not shown). The initial rates of the first step, calculated from the first three detectable levels of the lariat intermediate, were similar for DEAE II and DEAE II + I. The only effect of DEAE I on the first step was a shortening of the lag phase of the reaction (Fig. 2C). The first detectable levels of the lariat intermediate were observed at 60 min for DEAE II and at 40 min for DEAE II + I.


Figure 2: The activity in DEAE I affects the rate of the second step, but not the rate of the first step. A, a time course of in vitro splicing using the simultaneous assay described under ``Experimental Procedures.'' Splicing reactions were incubated for 20-180 min in the presence of 1 mM ATP and 5 mM creatine phosphate with DEAE II. B, a time course of in vitro splicing using the simultaneous assay described under ``Experimental Procedures.'' Splicing reactions were incubated for 20-180 min in the presence of 1 mM ATP and 5 mM creatine phosphate with DEAE I + II. Splicing intermediates and products were purified and visualized as described under ``Experimental Procedures.'' From top to bottom, icons indicate the lariat intermediate, lariat product, pre-mRNA, spliced product, and free 5`-exon. C and D, the data in A and B were quantified using a Molecular Dynamics PhosphorImager. The levels of the lariat intermediate, which was used to measure step I (C), and of the lariat product, which was used to measure step II (D), are plotted versus time.



The initial rate of the second step of splicing, calculated from the first three detectable levels of the lariat product, was markedly different for DEAE II versus DEAE II + I (Fig. 2D). The initial rate of the second step for DEAE II + I was 6.6-fold higher than the initial rate for DEAE II alone (Fig. 2D). There was also a broadening of the effect on the lag, with the first detectable lariat product appearing at 70 min for DEAE II versus 40 min for DEAE II + I (Fig. 2D). The fact that the increased lag was noted for both steps suggests that an early factor is present at lower concentration in the DEAE II fraction relative to nuclear extract. The results from two experiments are summarized in . Direct comparison between rates of steps I and II is not intended given that a conversion from signal strength (based on counts/minute) to moles of lariat product and intermediate has not been performed. Results from experiments using the sequential assay indicated that the magnitude of the effect on step II may be even greater than seen in (data not shown). These data indicated the existence of factor required primarily, if not exclusively, for the second step of splicing.

Characterization of the Second Step Activity

The DEAE I fraction was loaded onto CM-Sepharose to further purify the second step activity. Most, if not all, of the activity eluted in the flow-through/wash fractions of the CM-Sepharose column (data not shown). The properties of the activity for DEAE I and the CM-Sepharose flow-through/wash (CMFT) fractions were found to be identical (data not shown). The purification of this activity from nuclear extract was 5-fold. Other means of chromatographing this activity were tried, including other ion exchangers, dyes, a hydrophobic resin, and RNA-bound resins. In most of the cases, the activity was in the flow-through fraction, thus resulting in very poor purification (data not shown). The CMFT fraction was loaded onto Sephacryl S-300 HR to get an estimate of the size. The activity eluted in the void volume, indicating that it is very large, over 1.5 million Da.

The second step activity in the DEAE I and CMFT fractions was heat-sensitive. Treatment of DEAE I for 15 min at 37 °C did not affect second step activity, whereas treatment at 55 °C for 15 min resulted in an apparent defect in the second step (data not shown).

To evaluate the likelihood for a role of RNAs in this activity, the CMFT fraction was subjected to micrococcal nuclease treatment. Mock micrococcal nuclease treatment of nuclear extract had no effect on either step of splicing (Fig. 3, lane3). As expected, treatment with micrococcal nuclease abolished splicing activity (lane4). The micrococcal nuclease was inactivated before incubation with pre-mRNA as documented by lack of degradation of this RNA. DEAE II was only capable of carrying out the first step of the splicing reaction (lanes 5-8), but when supplemented with the untreated CMFT fraction, the lariat intermediate converted to the lariat product (lanes 9-11). The same was observed when the CMFT fraction was either mock-treated (lanes 12-14) or micrococcal nuclease-treated (lanes 15-17). These data suggest, but do not prove, that the second step activity does not depend on the integrity of an RNA.


Figure 3: The second step activity is resistant to micrococcal nuclease digestion. In vitro splicing was done using the sequential assay described under ``Experimental Procedures.'' Splicing reactions were incubated in the absence of ATP with nuclear extract (NE; lane1) or in the presence of 1 mM ATP and 5 mM creatine phosphate with nuclear extract (lane2), mock-digested nuclear extract (lane3), or nuclear extract digested with micrococcal nuclease (MN, lane4). Splicing reactions were incubated in the presence of 1 mM ATP and 5 mM creatine phosphate with DEAE II (lane5) and chased for 30-60 min with Buffer D (lanes 6-8), CMFT (lanes 9-11), mock-digested CMFT (lanes 12-14), or micrococcal nuclease-digested CMFT (lanes 15-17). Splicing intermediates and products were purified and visualized as described under ``Experimental Procedures.'' From top to bottom, icons indicate the lariat intermediate, lariat product, and pre-mRNA.



Northern analyses (26) were performed to determine which snRNPs were in the DEAE fractions. U2, U4, U6, and U5 snRNAs were found in the DEAE II and I fractions, whereas U1 snRNA was exclusively found in the DEAE II fraction (data not shown). Monomeric U2 snRNP, U5 snRNP, and the U4/U6-U5 tri-snRNP complex were detected only in the DEAE II fraction (data not shown). The U2, U4, U5, and U6 signals in DEAE I were all in a very large complex (data not shown). Therefore, DEAE II contained all of the required U snRNPs, making it unlikely that one of these is the step II activity. These data are consistent with the fact that DEAE II was competent to form spliceosomes (see below). It is important to note, however, that these data do not exclude the possibility that the activity is associated with U snRNPs, in particular a novel subpopulation of snRNPs.

The Second Step Activity Does Not Require ATP

We noted that the absence of exogenously added ATP had no effect on the activity of the CMFT fraction (data not shown). Moreover, a 5000-fold molar excess of the nonhydrolyzable ATP analogue AMP-PNP did not inhibit this activity. We investigated further whether or not ATP was required for the second step activity. To do this, we used the sequential assay described above and modified such that any ATP present during the first incubation was depleted before the chase (see ``Experimental Procedures''). The levels of ATP were followed by the addition of [-P]ATP to the reactions and detection by autoradiography of polyethyleneimine-cellulose TLC plates. High levels of ATP were detected after 70 min of incubation with DEAE II, even if no creatine phosphate was added (Fig. 4A, lanes1 and 2). In contrast to what is observed in yeast extracts (14) , glucose alone did not completely deplete ATP (lane3). Complete depletion of ATP required incubation with 25 µg/ml hexokinase and 12 mM glucose for 10 min (lane4). All detectable ATP was shown to be converted into ADP, AMP, and other products. Moreover, no ATP was regenerated during the chase with the DEAE I fraction (data not shown). Mock-depleted reactions and reactions incubated with 12 mM glucose alone, both of which had high levels of ATP, were used as positive controls for activity (Fig. 4B, lanes1, 2, 5, and 6). The depletion of ATP by hexokinase and glucose did not significantly affect the conversion of intermediates to products, which is most clearly seen for the lariat product (lanes3 and 4).


Figure 4: The second step activity does not require ATP. A, thin-layer chromatographic separation of ATP in splicing reactions was carried out as described under ``Experimental Procedures.'' Nucleotides were acid-extracted and chromatographed on polyethyleneimine-cellulose TLC plates developed with 1 M LiCl. Splicing reactions were incubated with DEAE II for 0 min (lane1) or for 70 min (lane2), with DEAE II for 70 min followed by a 10-min incubation with 12 mM glucose (lane3), or with DEAE II for 70 min followed by a 10-min incubation with 25 µg/ml hexokinase and 12 mM glucose (lane4) or a 10-min mock depletion (lane5). The chromatographic migration of ATP, ADP, and AMP is indicated. B, in vitro splicing was carried out using the sequential assay described under ``Experimental Procedures.'' Splicing reactions were incubated in the presence of 1 mM ATP with DEAE II for 70 min followed by a 10-min mock incubation and chasing with Buffer D (lane1) or DEAE I (lane2). Splicing reactions were incubated in the presence of 1 mM ATP with DEAE II for 70 min followed by a 10-min incubation with 25 µg/ml hexokinase (Hex.) and 12 mM glucose (Gluc.) and chased with Buffer D (lane3) or DEAE I (lane4). Splicing reactions were incubated in the presence of 1 mM ATP with DEAE II for 70 min followed by a 10-min incubation with 12 mM glucose and chased with Buffer D (lane5) or DEAE I (lane6). Splicing intermediates and products were purified and visualized as described under ``Experimental Procedures.'' From top to bottom, icons indicate the lariat intermediate, lariat product, pre-mRNA, spliced product, and free 5`-exon.



The Second Step Activity Can Complement Preassembled Spliceosome

Based on the fact that DEAE II was capable of catalyzing step I, we predicted that this fraction should be capable of spliceosome formation. This was assayed using native gel electrophoresis. Nuclear extract promoted the formation of two ATP-dependent splicing specific complexes: the pre-spliceosome (complex A) and the spliceosome (complex B) (Fig. 5A, lanes1 and 2). DEAE I was capable of forming complex A, but not complex B (lane3). DEAE II, not surprisingly, was capable of forming both (lane4), and the addition of DEAE I did not seem to enhance this activity (lane5). Thus, as expected, spliceosomes formed efficiently in the absence of the second step activity.


Figure 5: The second step activity can chase preformed spliceosomes. A, the second step activity was not required to form spliceosomes. Splicing complexes were formed by splicing reactions incubated in the absence of ATP with nuclear extract (NE; lane1) or in the presence of 1 mM ATP and 5 mM creatine phosphate with nuclear extract (lane2), DEAE I (lane3), DEAE II (lane4), or DEAE I + II (lane5). Splicing complexes were separated and visualized as described under ``Experimental Procedures.'' The splicing complexes spliceosome (B) and pre-spliceosome (A), -ATP complex (*), and nonspecific heterogeneous complexes (H) are indicated. B, a splicing reaction was incubated in the presence of 1 mM ATP and 5 mM creatine phosphate with DEAE II and subjected to velocity sedimentation on a 15%-30% glycerol gradient. RNAs found in fractions 6-10 are shown (lanes 1-5). Gradient fractions were chased with DEAE I for 70 min at 30 °C, and the RNAs found after the chase are shown (lanes 6-10). Splicing intermediates and products were purified and visualized as described under ``Experimental Procedures.'' From top to bottom, icons indicate the lariat intermediate, lariat product, pre-mRNA, spliced product, and free 5`-exon.



Functional spliceosomes were separated from pre-spliceosomes and commitment complexes by glycerol gradient velocity sedimentation (25) . On 15-30% glycerol (w/v) gradients, spliceosomes reproducibly sedimented in fractions 8-10. Spliceosomes assembled by incubation with DEAE II were sedimented on the same glycerol gradients. Fractions 8-10 were shown to contain free 5`-exon and lariat intermediate in addition to pre-mRNA, whereas all other fractions contained only pre-mRNA (Fig. 5B, lanes 1-5; data not shown). Therefore, the DEAE II spliceosomes could be separated after catalyzing the first step of splicing. The addition of DEAE I to the fractions resulted in progression through step II (lanes 6-10). These data suggest that the DEAE I activity can interact with preassembled spliceosomes. It is unlikely that all of the spliceosomes present in these fractions have undergone step I, but the observed levels of the lariat intermediate and product observed during the chase (lanes 6-10) argue that the DEAE I fraction may interact with spliceosomes that have already catalyzed the first step of splicing.

The Second Step Activity Can Be Purified Away from PSF

Since PSF has been suggested to have a role in step II (21) , we tested whether or not PSF was responsible for the second step activity in DEAE I. Nuclear extract was fractionated using poly(U)-agarose as described previously (20) . The flow-through/wash fraction (fraction I), which is equivalent to the PSF-depleted extract, had high levels of step II activity and no PSF (Fig. 6, A and B). The 1 M KCl fraction (fraction II) did not have any step II activity, but contained PSF (Fig. 6, A and B). Fraction III, which contains the splicing factor U2AF, had neither step II activity nor PSF (Fig. 6, A and B). Moreover, we showed that recombinant PSF did not have activity in our assay and that an antiserum to PSF did not inhibit this activity (data not shown). All of these data demonstrate that PSF is not a candidate for the novel step II activity in DEAE I.


Figure 6: A PSF-depleted fraction has step II activity. A, the poly(U)-agarose flow-through fraction (fraction I) has step II activity. In vitro splicing was done using the sequential assay described under ``Experimental Procedures.'' Splicing reactions were incubated in the presence of DEAE II for 70 min and then chased for 60 min with CMFT or fractions from nuclear extract fractionation through poly(U)-agarose (fractions I-III). The data were quantified using a Molecular Dynamics PhosphorImager. The step II activity was defined as the counts/minute in the lariat product in the tested fraction minus the counts/minute in the chase with Buffer D reaction (background). The numbers have been normalized to the number of counts/minute in each lane. B, Western analysis shows that PSF is only detected in fraction II. 7 µg of nuclear extract (NE) and fraction I and 3 µg of fractions II and III were run on a 12.5% SDS gel, transferred, and probed with an anti-PSF antiserum, which was detected by chemiluminescence as described under ``Experimental Procedures.''




DISCUSSION

In this report, we define an activity required for efficient catalysis of the second step of splicing. This activity is not required for the formation of splicing complexes or for the first transesterification reaction. Several results suggested that the activity is required at a step immediately preceding the second transesterification reaction. First, the lack of a requirement for ATP hydrolysis positions this activity late in the second step. ATP hydrolysis is required at several stages of the splicing reaction, including stages of the second step (Refs. 9, 12, 14, 18, 29, and 30; see also review in Ref. 2). The specific stage after step I at which ATP hydrolysis is required may be the PRP16-dependent conformational change observed in yeast extracts (11) . This change correlates with the 3`-splice site becoming inaccessible to oligonucleotide-directed cleavage by RNase H (11) and is probably linked to proofreading of the lariat intermediate and/or branch point sequences before proceeding to step II (31) . Assuming evolutionary conservation of these stages from yeast to humans, it is likely that our activity is required at a stage later than PRP16. Horowitz and Abelson (14) have proposed a similarly late role for PRP18. Their data and ours argue that the second transesterification reaction per se does not require ATP hydrolysis.

The second argument in favor of a late role for our activity is the ability to chase spliceosomes. Our interpretation of the data was that spliceosomes that had undergone step I (complex C1) were capable of functionally interacting with this activity and catalyzing step II. A similar late entry for PRP18 has also been suggested (14) . This suggests the possibility that the catalytic site for the second step is accessible to entry by factors acting late. It is formally possible that in complete nuclear extract, the activity may assemble early in spliceosome formation, but act late.

Of the well characterized yeast gene products required for step II, PRP18 is the best candidate as a homologue of our activity. Both share the lack of a requirement for ATP and similar chromatographic properties. In addition, we believe that our activity is required exclusively for step II, as is PRP18. This requirement for the DEAE I activity may not be absolute; the DEAE II fraction is capable of low levels of step II activity. Horowitz and Abelson (13, 14) showed that PRP18-depleted extracts can still carry on the second step of splicing, albeit at a much reduced rate. Moreover, a prp18 null mutant is viable at low temperatures, suggesting that PRP18 is not absolutely required for step II in vivo. Thus, PRP18 is a good candidate homologue of the DEAE I activity. We tested this relationship in two ways. Recombinant PRP18 did not substitute for the activity, and two anti-PRP18 antisera did not inhibit the DEAE I activity (data not shown). Neither of these two results permits a definitive conclusion, and thus, PRP18 remains a viable homologue for the DEAE I activity.

At this time, we have no evidence to suggest that a single polypeptide is responsible for this activity. In fact, based on the results of the Sephacryl S-300 HR column, it is possible that the activity is in a large complex. The only detectable U2, U4, U5 and U6 in the DEAE I fraction were observed in a large complex (data not shown). This complex may be related to the pseudospliceosomes described by Konarska and Sharp (33) . It is possible that the step II activity is associated with this complex; however, we do not favor a requirement for a snRNP because of the insensitivity to micrococcal nuclease.

Krainer and Maniatis (18) described two activities implicated in step II: SF3 and SF4A. SF3 was characterized as a micrococcal nuclease-resistant activity that was destroyed by heating at 45 °C for 10 min. Sawa et al.(30) showed that SF3 activity required ATP and was inhibited by nonhydrolyzable analogues of ATP. Thus, it is unlikely that our activity is SF3. SF4A was distinguished from SF3 by chromatography on spermine-agarose, and it is possible that our activity is the same as SF4A. Recently, Gozani et al. (19) showed that PSF was found among a group of proteins associated specifically with purified complex C1. Moreover, PSF-depleted extracts were shown to have splicing defects that suggested a role for PSF in step II (19) . This factor was originally described as a protein required for assembly of early splicing complexes (20) , and an early role for PSF has not been ruled out. The DEAE I activity is not required for complex A formation, which is clearly different than published for PSF. Also, we have shown that PSF is not responsible for our step II activity by separating PSF from the activity. Interestingly, anti-PRP18 antisera detect a human 54-kDa protein (p54 ) that, although seemingly unrelated to PRP18, is highly homologous to PSF (32) . PSF and p54 may be members of a family of factors with partially redundant activity analogous to SR proteins (34, 35, 36, 37) .

  
Table: Relative initial rates for steps I and II for DEAE fractions



FOOTNOTES

*
This work was supported in part by Grant GM49639 from the National Institutes of Health (to M. A. G.-B.).

§
Supported by a National Institutes of Health training grant through the Program in Cellular and Molecular Biology at Duke University.

Supported by the Cardiology Division, Department of Medicine, Duke University Medical Center.

**
To whom correspondence should be addressed: Dept. of Molecular Cancer Biology, Box 3686, Duke University Medical Center, Durham, NC 27710. Tel.: 919-613-8632; Fax: 919-613-8646.

The abbreviations used are: snRNP, small nuclear ribonucleoprotein; CMFT, CM-Sepharose flow-through; AMP-PNP, adenosine 5`-(,-imidotriphosphate).


ACKNOWLEDGEMENTS

We thank members of the Garcia-Blanco laboratory for helpful discussions. We are grateful to Drs. Adrian Krainer and David Horowitz (Cold Spring Harbor Laboratory) for PRP18 cDNA and anti-PRP18 sera and to Dr. Jim Patton for PSF cDNA and anti-PSF serum. We thank Sabina Sager for help in the preparation of this manuscript.


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