The Functional Role Of PRP28 Biology Essay

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This study focuses on the study of the functional role of PRP28, a DEAD-box protein that is essential for the assembly of spliceosome. In the consensus view, spliceosome is assembled on the pre-mRNA in a sequential manner that involves sequential association and dissociation of the different spliceosomal snRNPs and numerous non-snRNP splicing factors. The U1, U2, U5, and U4/U6 snRNPs are the major building blocks that form the core of the macromolecular machine. At the beginning of the splicing process, U1 snRNP is first recruited to the 5' splice site (5'ss) and other non-snRNP factors bind to the branch point sequence (BPS) and polypyrimidine tract of the pre-mRNA, forming the early (E) complex. U2 snRNP subsequently base pairs with the BPS in an ATP-dependent reaction to form the A complex. After the formation of A complex, RNP rearrangements occur at the BPS follow by the recruitment of the preassemble U4/U6.U5 tri-snRNP complex, forming the B complex. In order to activate the spliceosome, the snRNPs have to undergo major conformational and compositional rearrangements. During the process, U1 snRNP is displaced by U6 snRNP at the 5'ss and U4 snRNP is also destabilized and released from the complex, resulting in the catalytically active spliceosomal B' complex. The activated spliceosome then catalyze the first transesterification step of splicing, generating the C complex. This multistep assembly of spliceosome happens in an ATP-dependent manner and requires significant rearrangement and remodeling of the massive RNA-RNA, RNA-protein, and protein-protein interaction network, which are catalyzed by the highly conserved family of DExD/H-box RNA-dependent ATPase/helicases (reviewed in [1]). Although there is new hypothesis that spliceosome might come preassembled [2], it doesn't rule out that the activation of spliceosome requires the complex to undergo significant conformational and compositional rearrangement and the DExD/H-box family of RNA helicases play major roles in these processes.

(i) Work done by others

Biological roles of DExD/H-box proteins during different stages of splicing

DExD/H-box proteins were originally proposed to be RNA helicases [3] and were found to be involved in almost all RNA-related processes including splicing, mRNA export, translation and ribosome biogenesis (reviewed in [1]). The roles of DExD/H-box proteins in pre-mRNA splicing have become a major focus of functional studies of this protein family in the past decade. To date, eight evolutionarily conserved RNA helicases have been implicated in different steps of pre-mRNA splicing, likely for the resolution of RNA-RNA and RNA-protein interactions. In particular, DEAD-box proteins including Sub2/UAP56, Prp5, and Prp28 are implicated during the establishment of a functional spliceosome. On the other hand, DEAH-box proteins like Prp2, Prp16, Prp22, Prp43, and Brr2 are essential for the activation of transesterification reactions, mRNA release, and recycling of snRNPs (reviewed in [4-6]). These DExD/H-box proteins have been shown to associate with different snRNP complexes and carry out their distinct functions in ATP-dependent manner during the active remodeling of spliceosome.

PRP28 plays key role in spliceosome assembly

During the early stage of spliceosome assembly, U1 snRNP associates with the 5' splice site (5'ss) through base pairing between its snRNA and 5'ss region. This interaction is critical for the formation of the E-complex and continues until the start of B-complex formation. In the yeast system, various proteins including the U1snRNP proteins like U170K and U1C, and Cbp80p of the cap-binding complex (CBP) have been shown to be cross-linked to the 5'ss region, suggesting that they are part of the U1 snRNP-pre-mRNA complex during the formation of the commitment complex (same as mammalian E complex) {Zhang, 1999 #1}. These protein-RNA interactions are proposed to contribute to the stability between the RNA duplex between the U1 snRNA and 5'ss. Since the base-pairing between U1snRNA and 5'ss is replaced by that of U6snRNA:5'ss duplex during the formation of the pre-catalytic spliceosomal B complex, it is critical to reverse these protein-RNA interactions in order to unwind the 5 - 7 bp long U1snRNA:5'ss duplex. An essential member of the DExD/H-box proteins, Prp28, was suggested to promote the destabilization of these protein-RNA interactions as well as RNA-RNA interactions between the snRNA and 5'ss.

Prp28 in yeast (Prp28p) was initially identified as a component of the U4/U6.U5 tri-snRNP complex that is essential for the first step of splicing [8, 9]. Later studies found that both yeast and human Prp28 can be co-purified with U5snRNP and the latter was referred as U5-100kD [10]. Human Prp28 (PRP28) was shown to crosslink with the 5'ss through its ATPase domain during spliceosome assembly and throughout splicing {Ismaili, 2001 #15}. The formation of this interaction depends on NTP hydrolysis and is required to destabilize the base pairing of U1 snRNA with the 5'ss and facilitates the switch of 5'ss from U1 snRNA to U6 snRNA [11, 12]. Yeast genetic experiments demonstrated that the requirement for Prp28p in splicing could be bypassed by mutations in U1snRNA or U1snRNP component proteins, supporting the notion that Prp28p directly interacts with these factors and functions to destabilize the RNA-RNA and RNA-protein complexes at the 5'ss during the transition from the A complex to B complex, either directly or through modification of RNA structures [7, 13].

PRP28 is regulated by phosphorylation

To date, there is no evidence that either yeast or human Prp28 possesses any RNA unwinding activity in vitro, yet it was shown to inherit the predicted RNA-dependent ATPase activity [9]. It was reported that eIF4A, a closely related DEAD-box protein, requires the cofactor eIF4B for its optimal helicase activity in vitro [14]. Therefore, Prp28 might in fact do not possess any RNA helicase function, but it is also possible that the association/dissociation of an auxiliary factor or posttranslational modification is required to activate the helicase activity of Prp28. The activation of Prp28 might subsequently lead to alteration in the conformations of the RNPs and RNAs associate with it.

The N-terminal region preceding the helicase domain of human PRP28 contains several islands of arginine-serine (RS) dipeptides, which is the hallmark characteristic of the essential splicing factors SR proteins (reviewed in {Fu, 1995 #48}). The RS domains of SR proteins are extensively phosphorylated. Their phosphorylation states govern their roles in different pathways of RNA metabolism by modulating protein-protein and RNA-protein interactions (reviewed in [15, 16]). In addition to the RS dipeptides, PRP28 contains many arginine-aspartate (RD) and arginine-glutamate (RE) dipeptides that might mimic the phosphorylated state of RS dipeptides. These peculiar characteristics suggest that the N-terminal domain of PRP28 might play a role in its regulation during splicing. Indeed, a recent study has shown that human PRP28 undergoes phosphorylation by the splicing kinase SRPK2 and is critical for its association with the tri-snRNP complex [17]. More importantly, in addition to its essential role in the exchange of U6 for U1 snRNA at the 5'ss, immunodepletion and complementation studies demonstrated that phosphorylation of PRP28 is required for the integration of the tri-snRNP into the B complex. Since SRPK2 was also found to be associated with the tri-snRNP complex, the phosphorylation of PRP28 might have happened during or after the tri-snRNP assembly. Furthermore, PRP28 is likely to be integrated into the B complex with the tri-snRNP in its phosphorylated state. It will be important to determine if the phosphorylation of PRP28 will affect its ATPase or helicase activity.

(ii) Work done by us

Construction, expression and partial purification of different constructs of PRP28 for functional analysis

We have cloned full-length PRP28 into His-tag vector pET28a (EMD Biosciences). The construct was expressed with a N-terminal hexa-histidine tag in E. coli strain Rosetta2(DE3)pLysS (EMD Biosciences). The soluble fusion protein was first purified by anion-exchange and hydroxyapatite chromatographies, followed by affinity chromatography using Ni-NTA agarose. However, SDS-PAGE analysis of the elutant from the Ni-NTA chromatography showed the presence of non-specific proteins with lower molecular weight (Figure 1). Analysis of the elutant by western blotting using anti-His antibody showed that the non-specific proteins were N-terminal fragments of PRP28, likely the results of non-specific C-terminal degradation during protein expression and purification. Since PRP28 is an RNA-binding protein, we attempted to treat the partially purified protein with RNase A and benzonase to degrade non-specifically bound nucleic acids. However, the removal of nucleic acids resulted in protein precipitation and no soluble protein could be recovered. Therefore, only untreated protein was concentrated and flash-frozen for further analysis.

Since the RS-containing N-terminal domain of PRP28 is not conserved among different splicing helicases and is missing in its yeast ortholog Prp28p, we want to investigate its role in the regulation of PRP28 activity. We compared the amino acid sequences of human and yeast PRP28, along with those of other DEAD-box proteins like Vasa [18], DDX19 [19], and DDX3X [20], which three-dimensional structures were determined. We designed a new construct by deleting the first 373 amino acids of PRP28 to remove the entire non-homologous N-terminal domain. This new construct encodes the conserved helicase core of DEAD-box proteins, including all consensus motifs, and thus likely to retain its catalytic activity [6, 21]. The construct was cloned into the GST-tag vector pGEX4T2 and is referred to as GST-PRP28DN. GST-PRP28DN was first expressed in E. coli strain Rosetta2(DE3)pLysS and purified using hydroxyapatite chromatography followed by GST affinity chromatography (Figure 2). Like the full-length protein, the elutant from the GST affinity chromatography contained non-specific proteins with lower molecular weight. Analysis by western blotting using anti-GST antibody confirmed that the non-specific proteins were N-terminal fragments of GST-PRP28DN. Unlike the full-length construct, GST-PRP28DN remained soluble after nucleases treatment, likely the result of solubilization by the GST-domain and/or the absence of the N-terminal domain. Agarose gel electrophoresis revealed that the purified proteins were mostly nucleic acid free. Both nuclease-treated and untreated proteins were concentrated and flash-frozen for further analysis.

While the partially purified proteins are suitable for phosphorylation and binding studies to test the function of the N-terminal RS domain, proteins free of nucleic acids and of higher purity are required for further structural and functional studies. Therefore, our first objective is to purify both constructs of protein to homogenous and free of nucleic acids contamination.

Identification of phosphorylation region in PRP28

It was demonstrated that PRP28 is a potential target of SRPK2 (17). Members of the SRPK family are highly specific kinases that only phosphorylate serine residues within RS dipeptides {Wang, 1998 #47}. We therefore speculate that the phosphorylation of PRP28 occurs at its N-terminal domain. To test our hypothesis, we carried out steady state phosphorylation analysis using the partially purified full-length and N-terminal truncated PRP28 proteins. Free SRPK2 and His-PRP28, or GST-PRP28DN were incubated in the presence of 32P-ATP at 22oC respectively. Reactions were quenched by addition of 1X Laemmli buffer after 15 minutes and boiled at 98oC for 2 minutes subsequently. The reaction mixes were then resolved by SDS-PAGE. The gel was dried and exposed to autoradiography film (Figure 3). The result clearly showed that the full-length PRP28, but not GST-PRP28DN, was efficiently phosphorylated by SRPK2, confirming that the phosphorylation site(s) exists within the N-terminal domain of PRP28. Interestingly, we observed significant increase in background signal in the reaction of full-length PRP28. The strong background signal might be the result of excess retention of free 32P-ATP on the gel. However, this is unlikely because unhydrolyzed 32P-ATPs usually migrate much faster than proteins on a SDS-PAGE gel and rarely affect the background signal, which was indicated by the absence of background signal in the controls and GST-PRP28DN sample (Figure 3, lanes 1, 2, 3 and 5). Another possible explanation is that the ATPase activity of His-PRP28, which contain non-specifically bound nucleic acids, was enhanced upon phosphorylation by SRPK2, resulting in excess hydrolysis product 32P g-phosphates that migrate evenly on the SDS-PAGE gel. This hypothesis remains to be tested in our proposed studies.

Previous study on the biochemical activity of PRP28 was performed on its yeast ortholog Prp28p, which lacks the N-terminal RS domain [9]. Therefore the exact function and activity of human PRP28 remain to be resolved. Furthermore, the functional analyses of Prp28p were originally performed on fractions purified from S. cerevisiae. We cannot rule out the possibility that the presence of other co-purified endogenous proteins would have affected the results of the study. Therefore, the observed RNA-dependent ATPase activity of Prp28p and the absence of helicase activity remain to be proven definitely. Our second and third objectives are to investigate the RNA-binding, ATPase and helicase activity of purified recombinant human PRP28, and the effects of phosphorylation on these activities.


Objective 1: Purification of PRP28 to homogeneous for structural and functional studies.

Experimental design: We have shown that full-length His-tagged PRP28 and GST-tagged PRP28DN can be partially purified. In order to study the biochemical activities of PRP28 definitely, we shall purify the different constructs of protein to homogeneous. To isolate non-degraded protein products, we shall create new constructs of PRP28 by engineering a duel GST/His tag vector. This can be accomplished by modifying the E.coli T7 expression vector pET28b, which contains a His-tag at both ends of the restriction cloning region, to include an N-terminal GST-tag in place of the His-tag. Recombinant proteins expressed by E. coli will first be treated with nucleases to remove non-specifically bound nucleic acids. If proteins become insoluble, we shall use different types and/or combinations of detergent to resolublize the proteins. Alternatively, the proteins can be purified as inclusion bodies and renatured through dialysis. The proteins will be purified using various chromatography techniques until homogenous. The homogeneity of proteins will be confirmed by gel-filtration and dynamic light scattering.

Generation of duel-tagged PRP28 constructs: cDNA of GST containing a thrombin cut site at the C-terminus will be amplified by PCR using primers containing NcoI and BamHI restriction sites. The purified PCR products will be digested with NcoI and BamHI and subcloned into the NcoI-BamHI sites of the E.coli expression vector pET28b vector (EMD Biosciences) to replace the N-terminal His-tag and T7-tag. The cDNAs of full-length and N-terminal truncated PRP28 will then be cloned into pET28a-GST vector. The resulting constructs will have a GST-tag at the N-terminus and His-tag at the C-terminus.

Expression and purification of recombinant PRP28: Full-length and truncation mutant of PRP28 will be expressed as GST-His-fusion proteins in Rosetta2(DE3)pLysS cells (EMD Biosciences). To improve protein solubility, cells will be grown at 30oC until reaching an OD600 of 0.5, then induced with 0.1mM IPTG at 18oC overnight. Cells will be lysed by sonication. The cell lysate will be treated with RNaseA and benzonase at room temperature for 4 hours to remove non-specifically bound nucleic acids. In case of protein insolubility, protein pellet will be treated with 1% sarkosyl, 2% Triton X-100, and 20mM CHAPS to solubilize the protein [22]. The GST-His-fusion proteins will be purified by ion-exchange chromatography (GE Healthcare) and hydroxyapatite chromatography (Bio-Rad), followed by affinity chromatography using GST agarose (Qiagen) and subsequently Ni-NTA agarose (Qiagen). The GST-tag can be removed by incubation with thrombin followed by another step of GST-affinity purification. The proteins still containing the His-tag will then be loaded onto size-exclusion column (GE Healthcare) for the final step of purification. To ensure the purified proteins are RNA- and nuclease-free, samples untreated or after incubation with RNA and DNA markers will be analyzed by agarose gel electrophoresis.

Inclusion body purification and renaturation of proteins: Alternatively, if the proteins cannot be solubilized using detergents after nuclease treatment, inclusion bodies will be denatured by buffer containing 6M urea. The denatured proteins will first be purified by Ni-NTA agarose. The purified samples will be dialyzed three times against the renaturation buffer for 6 hours each. The refolded proteins will then be purified by GST-agarose, followed by size-exclusion chromatography.

Objective 2: To investigate the RNA-binding, ATPase and helicase activities of purified recombinant human PRP28 and their dependencies on the N-terminal region

Experimental design: PRP28 was shown to crosslink with the 5'ss through its ATPase domain in the presence of U4/U6.U5 tri-snRNP{Ismaili, 2001 #15}. Yet, it has not been studied if PRP28 alone is capable of binding to RNA. Furthermore, the RS domain presents at its N-terminal region is also well known for mediating RNA-protein interactions but its function in PRP28 is unknown. We want to investigate if apo PRP28 will interact with the 5'ss. If so, we shall also determine the role of the N-terminal RS domain RNA-binding. We shall use 5'ss sequence described by Ismaili et al to perform electrophoretic mobility shift assay (EMSA) to test the binding of the wild-type and N-terminal truncated proteins in the presence and absence of ATP and its non-hydrolyzable analog AMP-PNP. It was previously demostrated that Prp28p possesses RNA-dependent ATPase activity but no helicase activity [9]. However, similar biochemical studies have not been performed on human PRP28. We shall perform ATPase activity assays using the purified recombinant proteins to reconfirm the previous result obtained from study on the yeast ortholog. RNA unwinding assays will also be performed to determine if human PRP28 possesses any helicase activity. N-terminal truncated PRP28 will also be tested in the above assays to determine the role of the N-terminal region in ATPase or helicase activities of PRP28.

Electrophoretic mobility shift assays:

RNA aptamer of sequence 5'-AAG/GUAAGUAC-3' (bold letter indicates exon, / indicates exon/introns junction) will be 5'end labeled with 32P {Ismaili, 2001 #15}. Briefly, the apatamers will be dephosphorylated by calf intestinal phosphatase (CIP; Promega), follow by extraction with TRIzol reagent (Invitrogen) and ethanol precipitation. Dried RNA will be resuspended in buffer and phosphorylated by T4 polynucleotide kinase using 32P-ATP. To measure protein-RNA affinity, we shall use a constant RNA concentration and varied protein concentration. The RNA and protein will be incubated in buffer containing RNAaseIN (RNAse inhibitor, Promega) in the absence or presence of ATP or AMP-PNP. Products will be mixed with loading buffer, load onto nondenaturing polyacrylamide gels and separated by electrophoresis at 4oC. Data will be quantified with a Typhoon Phosphoimager.

RNA-dependent ATPase activity assays: ATPase activity of PRP28 will be determined by measuring the inorganic phosphate released during ATP hydrolysis using a direct colorimetric assay as described previously [23, 24, 25]. ATPase assay will be carried out in buffer containing Mg2+, DTT, various concentrations of whole yeast RNA (Type III, Sigma), RNAseIN and ATP. The ATPase reactions will be incubated at 37oC for various times. After incubation, malachite green-molybdenum reagent will be added the reaction mixture and incubate further at room temperature for 5 minutes. The reactions will be quenched with the addition of ethylenediaminetetraacetic acid (EDTA). The absorption at 630 nM will be measured for subsequent calculations of reaction rates. We shall also perform control reactions in the absence of RNA to determine the background hydrolysis of ATP and in the absence of ATP to monitor RNA degradation.

ATP-dependent helicase assays: Helicase assays will be performed following the protocol described by Strauss et al. with slight modifications [9]. In brief, RNA single strands will be prepared by in vitro transcription of pSP65 (Promega)(cut with XbaI) by SP6 RNA polymerase in the presence of rNTPs and [a-32P]UTP, and transcription of the pGEM1 plasmid (cut with PvuIII in the presence of rNTPs and cold UTP. Unincorporated radiolabel will be removed by gel filtration on Sephadex G-50 (GE Healthcare). The quality of radiolabeled RNA will be determined by SDS-PAGE and autoradiography. The partially complementary RNA duplex will be hybridized by heating up the mixture of radiolabeled and cold RNA to 95oC, followed by step-wise cooling to 4oC .

RNA helicase reactions will be carried out in the ATPase buffer at 37oC for various times. The products will be separated by polyacrylamide gel electrophoresis under non-denaturing conditions. The corresponding bands will be quantified with a Typhoon Phosphoimager.

Objective 3: To identify the site of phosphorylation and to determine the effect of phosphorylation of PRP28 on its RNA-binding, ATPase and helicase activities

Experimental design: After the determination of the RNA-binding, ATPase and helicase activity of PRP28, we shall test if the phosphorylation of PRP28 affects its biochemical activity. We shall first perform steady state phosphorylation analyses to quantify the number of phosphate(s) incorporated in PRP28 by SRPK2 using scintillation counting. The exact site(s) of phosphorylation will be determined by MALDI-TOF analyses. To confirm the result and to examine the role of phosphorylation in PRP28 activity, the identified site(s) will be mutated to alanine or glutamate to mimic the unphosphorylated and phosphorylated states of PRP28 respectively. The RNA-binding, ATPase and helicase activities of the wild type and mutant PRP28 will then be determined with or without phosphorylation by SRPK2. The serine-to-alanine mutant will serve as the negative control in these experiments. To ensure that any observed changes in PRP28's activity upon SRPK2 phosphorylation is not the result of ATP hydrolysis activity of residual SRPK2, the serine-to-glutamate mutant will serve as a mimic of the phosphorylated form of PRP28 and be tested in the absence of SRPK2.

Site-directed mutagenesis: All mutations or phosphorylation site(s) in PRP28 will be generated by single step polymerase chain reaction (PCR) using the QuickChange site-directed mutagenesis kit and relevant primers (Stratagene). All PRP28 mutants will be expressed and purified as described for the wild type protein.

Kinase activity assay and quantitation of phosphorylated sites: Free SRPK2 and different constructs of PRP28 will be incubated in the presence of 32P-ATP at 22oC. Reactions will be quenched by addition of 1X Laemmli buffer and subsequent boiling. Reaction mixes will be resolved by SDS-PAGE. The gel will be dried and exposed to autoradiography film. To quantitate the extent of phosphorylation, the dried radioactive bands will be excised and analyzed by a liquid scintillation counter.

MALDO-TOF analyses: MALDI-TOF analyses will be carried out by the laboratory of Dr. Majid Ghassemian, our existing collaborator from the Department of Chemistry and Biochemistry at the University of California San Diego. In brief, the analyses will be carried out using an ABI Voyager DE-STR spectrometer. PRP28 will be incubated with SRPK2 and ATP in the presence of Mg2+ for 30 mins at 22oC. Reactions will then be quenched with 5% acetic acid, desalted with Zip-tip C18 (Millipore), and eluted with 80% acetonitrile and 2% acetic acid for MALDI-TOF analysis. Unphosphorylated PRP28 control will be prepared in the same manner without ATP. Samples prepared in our laboratories will then be sent to Dr. Ghassemian's laboratory for analyses.

Phosphorylation of PRP28 by SRPK2 prior to activity assays: Free GST-tagged SRPK2 and His-tagged wild type or point mutants of PRP28 will be incubated in the presence of ATP and Mg2+at 22oC for 30 minutes. Excess free ATP will be removed using Zeba Micro Spin desalting columns (Thermo Scientific). The reaction mixture will then be incubated with GST-resin to bind and remove the free GST-SRPK2. The supernatant that contains PRP28 only will be used for subsequent activity assays.

Milestone table for experimental work








Construction, expression and purification of wild type and mutant proteins





ATPase and helicase activity assays



Phosphorylation studies of PRP28


Activity assays testing the effects of phosphorylation