PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome

Abstract

The assembly of the spliceosome is an ATP-dependent process. The splicing factor PRP16 contains variations of several motifs that define the eIF-4A-like ATP-dependent RNA helicase family. The protein has now been purified and shown to exhibit RNA-dependent ATPase activity. PRP16 is required specifically for the second catalytic step of the splicing reaction in vitro. This function requires ATP binding and/or hydrolysis, which appears to be concomitant with release of the protein from the spliceosome. PRP16 may be the prototype for a set of splicing factors which use ATP to drive a cycle of conformational changes.

Effects of oligonucleotide length and atomic composition on stimulation of the ATPase activity of translation initiation factor eIF4A

“…An analogous strategy was used to determine the value of K m oligo for U 15 RNA with a (P-S) backbone [abbreviated U 15:29-OH,(P-S) ]+ Stimulation provided by 2) to yield K m oligo ϭ 13 mM for the phosphodiester oligonucleotide and K m oligo , 0+25 mM for the phosphorothioate oligonucleotide+ B: ATPase activity with subsaturating ATP•Mg (1 mM), 0+25 mM eIF4A and subsaturating U 20 RNA (3 mM) is inhibited by addition of U 20 DNA with a phosphodiester (Ⅲ ) or a phosphorothioate backbone (▫ )+ Normalized values were obtained by dividing the observed rate constants by the rate constant measured in the absence of inhibitory oligonucleotide+ Curves represent fits to simple inhibition model (Equation 5), yielding K i oligo ϭ 12 mM for the phosphodiester oligonucleotide and K i oligo , 0+25 mM for the phosphorothioate oligonucleotide+ U 15:29-OH,(P-S) was measured in the presence of an inhibitory oligonucleotide, U 20:29-H,(P-S) , which was included in the reaction to compete with U 15:29-OH,(P-S) for binding to eIF4A and thus increase K m oligo,app (Scheme 1)+ Figure 4B shows the effect of this inhibitor on the U 15:29-OH,(P-S) concentration dependence for ATP stimulation, giving observed K m oligo,app values of 5+7 and 11 mM with 1 and 2+5 mM U 20:29-H,(P-S) present+ With these K m oligo,app values and the value of K i oligo for U 20:29-H,(P-S) (24 nM, determined from Fig+ 4A), K m oligo for U 15:29-OH,(P-S) was calculated to be 0+12 mM according to Equation 1c+ K m oligo for this (P-S)-containing oligonucleotide is ;100-fold smaller than K m oligo for the analogous (P-O)-containing oligonucleotide (13 mM, Fig+ 3A)+ Another DEAD box protein, PRP16, also binds tightly to phosphorothioate oligonucleotides Given the unexpectedly strong interaction between eIF4A and phosphorothioate-substituted oligonucleotides, it was of interest to determine if other DEAD box proteins also bind (P-S)-containing oligonucleotides tightly+ The DEAD box protein Prp16 is an RNAdependent ATPase involved in pre-mRNA splicing (Schwer & Guthrie, 1991;Wang & Guthrie, 1998)+ U 20:29-H,(P-S) inhibited the ability of poly(U) RNA to stimulate Prp16's ATPase activity .3,000-fold more than the analogous (P-O)-containing oligonucleotide (data not shown)+ The rate constant measured for Prp16 saturated with poly(U) RNA was not affected by the presence of the inhibitory oligonucleotide, consistent with competitive inhibition+ Thus, Prp16, like eIF4A, appears to bind (P-S)-containing oligonucleotides considerably more tightly than (P-O)-containing oligonucleotides+ Dependence of oligonucleotide affinity on the number and position of phosphorothioate linkages within an oligonucleotide Inhibition constants were measured for oligo(U) DNA of varying lengths with (P-O) or (P-S) backbones (Table 1)+ For both (P-O)-and (P-S)-containing oligonucleotides, affinity increases as length is increased+ The ratio of the inhibition constants for (P-O) and (P-S) oligonucleotides of the same length isolates the effect of substituting (P-O) linkages with (P-S) linkages from the overall effect of changing oligonucleotide length (ratio, We also measured the inhibition constants for a series of U 20 DNA oligonucleotides containing a varying number of (P-S) linkages in an otherwise (P-O) backbone (Fig+ 5)+ As the number of (P-S) linkages increases, there is an increase in affinity, with an effect of four-to eightfold per added block of four or five (P-S) linkages+ In addition, the inhibition constants for different oligonucleotides containing equal numbers of (P-S) linkages are the same within error, suggesting that (P-S) linkages at different locations within the oligonucleotides provide similar incr...…”

The carboxy terminal WD domain of the pre-mRNA splicing factor Prp17p is critical for function

“…In yeast, the second-step factors have been ordered with respect to an ATP-dependent event during the second step (Schwer & Guthrie, 1991;Horowitz & Abelson, 1993;Ansari & Schwer, 1995;Jones et al+, 1995)+ The RNA helicase Prp16p hydrolyzes ATP and is required for a conformational change that occurs in the spliceosome after the first step (Schwer & Guthrie, 1992)+ yPrp17p is required before or during this event; Prp18p and Slu7p are required afterwards (Umen & Guthrie, 1995b)+ A role for the yeast Prp22 in the second step of splicing has been shown more recently (Schwer & Gross, 1998), and it is proposed to act in conjunction with Slu7 and Prp18+ Rearrangements in the spliceosome after the first step are required to identify and position the 39 splice site for the second step+ After the first step of splicing, the highly conserved loop I of U5 snRNA plays an important role in aligning the two exons for the second-step catalytic site+ In yeast, this loop is essential for the second step; however, in humans it is not required (O'Keefe et al+, 1996;Segault et al+, 1999)+ The loop I uridines are likely involved in noncanonical base pairing with the exon sequences near the splice sites, which are weakly conserved compared to elements in the intron+ Additional interactions appear to be necessary for stabilization of the U5 snRNA and the two exons during the second step+ For example, the highly conserved U5 snRNP protein Prp8p crosslinks to both the 59 and 39 splice sites (Wyatt et al+, 1992;Teigelkamp et al+, 1995;Umen & Guthrie, 1995a, 1995b)+ In addition, Slu7p and yPrp17p have been linked genetically to the function of U5 snRNA loop I Seshadri et al+, 1996)+ Prp18p and Slu7p have been shown to physically interact (Zhang & Schwer, 1997); however, no other direct interactions have been shown between proteins required for the second step+ Studies of synthetic lethality suggest that there is a functional interaction between all the second-step factors (reviewed in Umen & Guthrie, 1995c)+ Alleles of PRP17 are synthetically lethal with alleles of SLU7, PRP16, PRP18, PRP8, SLT11, U5 snRNA, and U2 snRNA Umen & Guthrie, 1995a;Seshadri et al+, 1996;Xu et al+, 1998), implying specific, and possibly direct interactions+ Insufficient knowledge of the proteins and/or RNAs that interact with yPrp17p, and the function of this protein, severely limits our understanding of its role in the second step of pre-mRNA splicing+ Through co...…”

Influenza virus NS1 protein alters the subnuclear localization of cellular splicing components

“…From the loss of the pre-mRNA specific protection band and the increase in the accumulation of the intron-plus-second exon protection band, it could be suggested that NS l protein enhances the formation of the spliceosomal complex but inhibits the second step in the splicing reaction. Thus, it could interfere with the function of the U2, U4/U6 and/or the U5 snRNPs which may be involved in the catalytic steps of splicing (Newman & Norman, 1992) or with the activity of the mammalian counterpart of the PRP16 factor, a non-snRNP yeast protein with RNAdependent ATPase activity acting in the spliceosome before the second splicing step (Schwer & Guthrie, 1991).…”