- Email: [email protected]
[17]In vitro assays for characterization of RNA-protein complexes involved in pro-opiomelanocortin mRNA translation
[17]
In Vitro Assays for Characterization
of RNA-Protein Complexes Involved in Pro-opiomelanocortin mRNA Translation Stephanie D. Flagg, Corinne M. Spencer, and James H. Eberwine
Introduction Proper regulation of pro-opiomelanocortin (POMC) gene expression is critical to the mammalian stress response. In response to physical or psychological stress, the hypothalamus secretes vasopressin and corticotropin-releasing hormone (CRH), which in turn stimulate POMC gene expression in the anterior and intermediate lobes of the pituitary. This stimulation results in the increased synthesis of POMC-derived peptides including adrenocorticotropin (ACTH) and/3-endorphin. Adrenocorticotropin then stimulates cortisol synthesis and secretion in the adrenal cortex. Glucocorticoids negatively regulate POMC gene expression in the anterior pituitary, while dopamine assumes this role in the intermediate lobe. Several groups have noted a discordance in the time courses over which POMC peptide and mRNA levels are altered in response to alterations in stress hormones ( 1 - 3). In pulse-chase experiments, Shiomi et al. (4) have noted a 50% increase in the efficiency of POMC peptide synthesis in rats exposed to acute stress at a time when no change in POMC mRNA levels is detected. Additionally, we have observed a change in the polysome distribution of POMC mRNA in response to treatment of AtT-2.0 cells (a mouse pituitary tumor) with dexamethasone or forskolin, a compound that mimicks the action of CRH (5). These findings suggest that POMC gene expression is regulated at the level of translation. Translational control refers to the regulation of any of several factors that determine the efficiency of protein synthesis. These factors include the rate at which ribosomal binding and initiation of translation occur, the rate at which each ribosome translocates along a mRNA molecule, and the fraction of mRNA that is actively being translated in polysomes. In addition, several features of a mRNA transcript are thought to influence its translation, including the exposure of the 7-methylguanosine cap, the context of the initiator methionine, and the degree of secondary and tertiary structure present within the mRNA (6). Control of gene expression at the level of translation allows for protein levels to be rapidly altered without necessitating a change in steady state mRNA transcript levels, thereby bypassing the time delay that is associated with RNA transcription, processing, and transport. The added fact that translation is an amplification process in which one mRNA molecule can be translated into thousands of molecules of proMethods in Molecular Genetics, Volume 8
Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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tein product allows this rapid control mechanism to alter protein levels dramatically. Translational control is thus a mechanism by which cells may rapidly respond to changes in the environment. One commonly utilized mechanism by which translational control is effected is through the interaction of an RNA-binding protein with a specific RNA sequence or structure. The binding of such proteins can alter the stability of RNA structures formed by intramolecular base pairing, which in turn can influence the efficiency by which a mRNA is translated. Protein-RNA interactions have been shown to impede the translation of many prokaryotic genes (7, 8), and have been demonstrated to play an important role in the expression of several eukaryotic genes as well (9, 10). Such protein-RNA interactions may play a role in the translational regulation of POMC gene expression. Computer modeling of POMC mRNA predicts the presence of a stem-loop structure of - 4 5 kcal/mol stability within the 5' coding region (Fig. 1). Data from in situ transcription experiments suggest that this predicted stemloop sequence exists in situ in rat pituitary tissue (5). On the basis of these data, we hypothesized that cytoplasmic proteins in rat pituitary and AtT-20 cells might recognize and bind to the stem-loop structure in POMC mRNA to effect its translational control. With the use of in vitro assays we have demonstrated the plausibility of this hypothesis. Cytoplasmic extracts from rat pituitary and AtT-20, but not COS cells (a monkey kidney fibroblast cell line), impede the translation of POMC mRNA in an in vitro translation assay (11). We have characterized RNA-protein interactions that play a role in POMC translation with the use of RNA gel mobility shift and ultraviolet (UV) cross-linking assays. These techniques are the focus of this chapter and are useful tools in the study of RNA-protein interactions that assume roles in RNA translation as well as those involved in RNA processing or transport.
In Vitro T r a n s c r i p t i o n
DNA sequences encoding the RNA sequence of interest should be cloned into a vector downstream of a strong bacteriophage RNA polymerase promoter such as T7, T3, or SP6. To obtain capped, full-length POMC mRNA that can be readily translated in rabbit reticulocyte lysates, we have used linearized DNA template and T7 RNA polymerase with the protocol for the mMessage mMachine in vitro transcription kit (Ambion, Inc.). For the production of large quantities of nonradioactive competitor RNAs, we have achieved optimal success with the use of the appropriate (T7, T3, or SP6) MEGAscript kit using the unmodified procedure as outlined by the manufacturer (Ambion, Inc.) or by using a high concentration of T7 RNA polymerase (1000 U//zl; Epicentre Technologies) as described below: 1. DNA template must be free of RNases and thus should be purified by cesium chloride gradient centrifugation rather than by commercially available column pro-
[17]
PROTEINS INVOLVED IN POMC mRNA TRANSLATION
AC C G C G A A C-G U-A C- G~-'~-- + 120 C "A A A G-C G-C A-U C-G C
323
(RAT=A)
Gu i G-C A-U C-G C-G G-C A-U
U C-G G-C A A G-C G-C UoG C-G C-G G-C
5'
3'
FIG. 1 Computer-predicted secondary structure of mouse POMC RNA stem-loop. The rat POMC stem-loop is identical to that of the mouse, with the exception of a single base pair substitution (A for G at position 120).
cedures, which rely on RNase treatment to remove contaminating RNA. DNA template should be linearized with the restriction enzyme that cuts 3' to the insert so as to minimize the amount of vector sequences transcribed and to ensure that all RNA transcripts synthesized will be of the same length. Restriction enzyme-cut plasmid DNA is extracted with equal volumes of phenol-chloroform and precipitated with a • vol of 3 M sodium acetate, pH 5.2, and 2.5 vol of ethanol. The pellet should be resuspended in Tris-EDTA (TE) buffer that has been filter sterilized. Because they stick to the nitrocellulose membrane, RNases can be removed from solutions during the filtration process. 2. Set up in vitro transcription reactions in RNase-free microfuge tubes. All buff-
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ers are made RNase free by sterile filtration or by treatment of buffers that do not contain primary amines with diethyl pyrocarbonate (DEPC). H20 Transcription buffer (5 • 200 mM Tris (pH 7.5), 35 mM MgC12, 50 mM NaC1, 10 mM spermidine Dithiothreitol (DTT, 100 mM) RNasin (40 U; Promega, Madison, WI) ATP (10 mM) CTP (10 mM) GTP (10 mM) UTP (10 mM) Linearized template DNA T7 RNA polymerase (1000 U; Epicentre Technologies)
43/zl 20/zl 10/xl
1/xl 5 #l 5/xl 5/xl 5/zl 5/xl = 1/xg
1/xl
3. Incubate the reactions for 90 min at 37 ~C. 4. Remove the DNA template by addition of 2/zl of RNase-free DNase I (10 U/ /zl; BMB) and continue the incubation at 37~ for 30 min. 5. Extract the RNA with phenol-chloroform, and ethanol precipitate. 6. Resuspend the RNA in 10 #1 of sterile-filtered H20 and drop dialyze the sample on nitrocellulose filters (Cat. No. VSWP01300; Millipore, Bedford, MA) for 1-4 hr to remove free triphosphates. Repeat the phenol-chloroform extraction and ethanol precipitation. 7. Obtain a spectrophotometric reading of sample at 260 nm to quantitate the RNA. Radiolabeled riboprobe can be synthesized by the above protocol if 30/xCi of [ce-32p]CTP is added to the reaction tubes and the final concentration of unlabeled CTP is reduced to 0.5/xM. T7 RNA polymerase of a lower specific activity (GIBCOBRL, Gaithersburg, MD) may be employed in riboprobe synthesis if a smaller quantity of RNA needs to be produced. Additionally, free triphosphates can be removed with two sequential ethanol precipitations. Counts per minute are determined by scintillation counting.
Cytoplasmic Extract Preparation S 100 Cytoplasmic extracts are prepared as described (12). Briefly: 1. Grow cells in culture to 5 • 105 and harvest by centrifugation for 10 min at 2000 rpm in a Sorvall HG4L rotor. All subsequent steps should be performed at 4 ~C. 2. Resuspend the pellet in 5 vol of phosphate-buffered saline.
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PROTEINS INVOLVED IN POMC mRNA TRANSLATION
325
3. Centrifuge as in step 1. 4. Resuspend in five packed cell pellet volumes of buffer A [10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES, pH 7.9 at 4~ 1.5 mM MgCI2, 10 mM KC1, 0.5 mM DTT] and incubate for 10 min on ice. 5. Centrifuge as in step 1. 6. Resuspend the pellet in 2 vol of buffer A, and lyse cells with at least 10 strokes of an all-glass Kontes Dunce homogenizer, type B pestle. The homogenate should be checked at this point for the efficiency of cell lysis. For effective lysis of AtT-20 cells, an additional one or two rounds of homogenization are required. 7. Centrifuge as in step 1 to pellet nuclei. 8. Mix the resulting supernatant with 0.11 vol of buffer B [300 mM HEPES (pH 7.9 at 4~ 1.4 M KC1, 30 mM MgC12] and centrifuge for 60 min at 100,000 gav. 9. Dialyze the supernatant for 5 to 8 hr against 20 vol of buffer D [20 mM HEPES (pH 7.9 at 4~ 100 mM KC1, 0.2 mM EDTA, 20% (v/v) glycerol, 0.1 M KC1, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5 mM DTT]. Protein determinations can be made by the use of the Bradford assay (Bio-Rad, Richmond, CA). Aliquot cytoplasmic extracts and store at - 8 0 ~C. Note: Dithiothreitol and PMSF should be added to buffers at the time of use.
In Vitro T r a n s l a t i o n s
We have used a rabbit reticulocyte lysate system (Amersham, Arlington Heights, IL) to demonstrate the ability of protein-RNA interactions to regulate the in vitro translation of POMC mRNA (Fig. 2). Reaction conditions are outlined by the manufacturer. Briefly, a 10-/zl reaction is set up consisting of 200 ng of the full-length mRNA of interest (or control RNA such as brome mosaic virus; Amersham), 10 #Ci of L-[35S]methionine (translation grade; Amersham), 4 /zl of cytoplasmic extract or Dignam buffer D (see cytoplasmic extract preparation), and 4 #1 of nuclease-treated, message-dependent rabbit reticulocyte lysate. Translation reactions are incubated for 1 hr (time course should be empirically determined) at 30~ and then mixed with 2• sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, boiled for 5 min, and electrophoresed on the percentage denaturing polyacrylamide gels that will achieve effective separation of the translated protein product. After treatment with Entensify universal autoradiography enhancer (NEN-Dupont, Boston, MA), gels are dried and exposed to film for 3 - 7 days at -80~ Competitor RNAs can be included in the translation reactions to evaluate the ability of an RNA sequence or structure to compete with the full-length mRNA for interaction with cytoplasmic protein and attenuate the ability of cytoplasmic extract to alter the translation of the full-length mRNA. Certain issues must be kept in mind in performing these experiments and in interpreting the resulting data. High concentrations of RNA inhibit protein synthesis in rabbit reticulocyte lysates, perhaps by sequestering initia-
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lxg AtT20 Extract
6.4
0
1.6
3.2
200 ng POMC RNA
-
+
+
+ -- 221.2 kDa
-- 105.7 kDa
-- 74.7 kDa
-- 46.3 kDa
"- 27.6 kDa
"- 18.6 kDa
FIG. 2 The translation of P O M C RNA in vitro is inhibited by AtT-20 cytoplasmic extract. Two hundred nanograms of capped full-length P O M C m R N A was translated in a rabbit reticulocyte lysate system in the presence of increasing amounts of AtT-20 cytoplasmic extract (lanes 2 - 4 ) . The first lane is a control reaction in which no RNA was added to the reticulocyte lysate. The arrow indicates the position of the P O M C peptide as determined by immune precipitation (data not shown).
[17]
PROTEINS INVOLVED IN POMC mRNA TRANSLATION
327
tion and elongation factors (13). Furthermore, the addition of high concentrations (> 1 ~g/ml) of double-stranded RNA to reticulocyte lysate can lead to a generalized translational arrest (14, 15) by blocking the activation of a double stranded RNAdependent protein kinase that phosphorylates initiation factor eIF-2. Thus, proper controls are required to evaluate data from these experiments.
R N A Gel M o b i l i t y Shift A s s a y RNA gel mobility shift assays are performed essentially as described (16). 1. Set up RNA-protein binding reactions in RNase-free microfuge tubes with 5 25 #g of cytoplasmic extracts (or lesser amounts of purified protein), 40 U of RNasin (Promega), and 50,000 cpm of labeled riboprobe in 10 mM HEPES (pH 7.6), 3 mM MgCI2, 40 mM KC1, 5% (v/v) glycerol, and 1 mM DTT in a 20-/.~1 reaction volume. One reaction should be set up with riboprobe in the absence of protein extract to demonstrate the mobility of unretarded RNA transcript. 2. Incubate binding reactions at room temperature for 20 rain to allow proteins to interact with the riboprobe. Salt concentrations, incubation time, and temperature may need to be modified to optimize binding. . 3. Add 1 #1 of heparin (100 mg/ml; Sigma, St. Louis, MO) and continue the incubation at room temperature for 10 min. The addition of heparin should decrease the binding of proteins that interact nonspecifically with the phosphate backbone of the riboprobe. 4. Electrophoresis is performed as described (Ref. 17, with modifications). Load binding reactions directly onto a 5% (w/v) nondenaturing polyacrylamide (60: 1, acrylamide-bisacrylamide) gel that has been preelectrophoresed for 30-60 min at 8 V/cm in 45 mM Tris-borate, 1 mM EDTA. Electrophorese the gel for 2 to 2.5 hr at 8 V/cm. 5. Dry the gel and autoradiograph at room temperature for 12 to 24 hr. The RNA gel mobility shift assay is a rapid method that can be used to demonstrate whether a purified protein or proteins within an extract preparation can bind specifically to an RNA fragment. Control reactions performed in the absence of protein demonstrate the unretarded migration of riboprobe within the gel. The addition of cytoplasmic extract containing proteins that recognize the riboprobe results in the formation of riboprobe-protein complexes that are distinguishable by their slower migration through the gel (Fig. 3). Competition experiments can be performed by the inclusion of a 10- to 1000-fold molar excess of unlabeled competitor RNAs to the binding reactions (step 1). Unlabeled competitor RNAs that specifically compete with the riboprobe for protein binding will disrupt the formation of riboprobe-protein complexes (less riboprobe
328
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TRANSCRIPTION
AtT20 Extract
-
+
+
Sense Competitor RNA
.
.
+
Complex 2 Complex 1
Free Riboprobe
FIG. 3 RNA gel mobility shift assay with a 32p-labeled POMC stem-loop RNA in the absence (lane 1) or presence (lane 2) of 5/zg of cytoplasmic AtT-20 extract. Two RNA protein complexes are observed on the addition of AtT-20 cytoplasmic extract. These complexes are competed away by the addition of 100 ng of unlabeled POMC stem-loop RNA (lane 3).
will be shifted) to a greater degree than will cold nonspecific RNAs. Nonspecific RNAs that are commonly used in this assay include RNAs of the same length as the riboprobe that are synthesized from linearized vector DNA templates, tRNA, and poly(rI-rC). Proteinase K should be included in a control reaction to demonstrate unequivocally that the retarded migration of riboprobe is due to the interaction of protein with the RNA. The RNA gel mobility shift assay does not provide information on the molecular weight of the proteins that interact with the riboprobe, and does not indicate the number of proteins that contribute to the riboprobe-protein complexes formed. For this type of information, a UV cross-linking assay may be performed.
Ultraviolet Cross-Linking Assay Binding reactions are performed as described in steps 1 to 3 for the gel mobility shift assay, except that reactions are set up in the caps of microfuge tubes. Competition experiments can be performed by adding unlabeled competitor RNAs in the binding reactions as was described for the gel mobility shift assay. After the addition of heparin:
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PROTEINS INVOLVED IN POMC mRNA TRANSLATION
329
1. Binding reactions are exposed to UV light (Sylvania G15T8; GTE Sylvania, Inc.) for 10 min at a distance of 4.5 cm (18). A time course should be performed with the UV source at various distances to optimize conditions. 2. Incubate the reactions at room temperature with 5 /xg of RNase A to digest RNA not protected by covalent linkage to protein. Some protocols include a variety of RNases (T1, T2, and V1) in this incubation. RNase A cleaves after pyrimidine residues; RNase T1 cuts after guanosine residues; T2 cleaves after any residue; and RNase VI cleaves double-stranded RNA. The addition of these enzymes to the reactions may be important for the complete digestion of highly structured RNAs. 3. Add an equal volume of 2• SDS sample buffer to the reaction tubes, and boil the samples for 5 min before separating the proteins by electrophoresis on 8-10% (w/v) SDS-polyacrylamide gels according to a standard protocol (19). 4. Dry the gel and visualize complexes by autoradiography. Ultraviolet treatment of the binding reactions covalently cross-links to RNA proteins that are in direct association with it. RNase treatment of the RNA-protein complexes degrades all riboprobe that is not protected by direct cross-links to protein. When these samples are run on denaturing polyacrylamide gels, the proteins that are cross-linked to small fragments of riboprobe are separated by virtue of their molecular weight and can be visualized by autoradiography. This assay allows for the estimation of molecular weight of the RNA-binding proteins and gives information as to the number of proteins that can bind directly to the riboprobe (Fig. 4). This technique, however, has several limitations. A single band on a UV cross-linking experiment does not correspond necessarily to one unique RNA-binding protein because one band may represent either a complex of proteins (if two or more proteins become cross-linked by the UV treatment) or the partial degradation product of an RNA-binding protein. In addition, a protein that binds indirectly to the RNA via a protein-protein interaction may not become covalently cross-linked, and therefore would not be visualized on autoradiography.
In Vitro M u t a g e n e s i s o f R N A R e c o g n i t i o n Site The RNA-binding site recognized by extract proteins can be further delineated by site-directed mutagenesis of the RNA fragment. Mutant RNAs can be synthesized from mutagenized DNA templates and used as riboprobes or competitors in gel mobility shift and UV cross-linking assays. Additionally, mutant RNAs can be studied in in vitro translation assays and in vivo, after transfection of cells with reporter gene constructs. From these types of experiments, one can ascertain the degree to which sequence and structure of an RNA contribute to the RNA-protein interactions being studied. We have created mutants in the stem-loop structure of POMC mRNA with the use
330
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min UV
0
10
30
--221 kDa
-- 106 k D a
- 75 k D a
-- 4 6 k D a
FIG. 4 Ultraviolet cross-linking experiment with 32p-labeled POMC RNA stem-loop and 18.7 /xg of AtT-20 cytoplasmic extracts. Samples were exposed to UV light for 0, 10, or 30 minutes as indicated (lanes 1-3). Four RNA-protein complexes are observed.
of the Altered Sites in vitro mutagenesis system (Promega). This system requires subcloning the DNA encoding the RNA of interest into the multiple cloning site of the pSELECT-1 phagemid-plasmid, which contains SP6 and T7 promoters. Singlestranded DNA (ssDNA) is produced by infection of host bacterial cells with helper phage. This ssDNA is annealed to a 5'-phosphorylated mutant oligonucleotide and the synthesis of the mutant strand is completed with the addition of T4 DNA polymerase and T4 DNA ligase. The percentage of mutants that is created is optimized by performing a first round of transformation in BMH 71-18 MutS, a repair-defective Escherichia coli strain. A second round of transformation is performed in JM109 cells in order to ensure segregation of wild-type and mutant clones. Clones are sequenced to confirm the identity of mutants. The pSELECT-1 plasmid contains a gene for tetracycline resistance and a nonfunctional ampicillin resistance gene. This feature, and the provision of an oligonucleotide that when included in the annealing reaction results in the repair of the ampicillin resistance gene, allows for
[17] PROTEINSINVOLVEDIN POMC mRNATRANSLATION
331
ampicillin selection for mutant strand synthesis and increases the percentages of mutants obtained. A newer version of this system (Altered Sites II in vitro mutagenesis system; Promega) is currently available that boasts as advantages (a) the inclusion of a protocol for mutagenesis of double-stranded DNA and (b) the ability to perform multiple rounds of mutagenesis without resubcloning by alternating the knockout and repair of the tetracycline and ampicillin genes. We have found the ES 1301 MutS repairdefective E. coli strain that currently must be used for multiple rounds of mutagenesis to be difficult to transform. Competent ES 1301 MutS cells soon may be available from Promega.
Summary Although we have chosen to illustrate the utility of these techniques in the characterization of RNA-binding proteins that are involved in translation, these techniques are also useful in characterizing RNA-binding proteins that have other functional roles within the cell, such as the processing and transport of RNA. In particular, these techniques are being utilized to characterize RNA-binding proteins involved in the subcellular distribution of RNAs as well as those thought to be involved in regulating the course of various neurological diseases. Once the specificity of an RNA-protein interaction has been characterized by these methods, one can isolate the protein(s) involved by the use of a direct cloning approach via a Northwestern hybridization screen (20, 21) or by biochemical purification techniques that take advantage of RNA affinity chromatography (22, 23).
Acknowledgments J.H.E. is an established investigator of the American Heart Association. S.D.F. is supported by NIGMS and is a Dupont-Merck Scholar.
References 1. C. L. C. Chen, E T. Dionne, and J. L. Roberts, Proc. Natl. Acad. Sci. U.S.A. 80, 2211 (1983). 2. N. C. Birnberg, J.-C. Lissitzky, M. Hinman, and E. Herbert, Proc. Natl. Acad. Sci. U.S.A. 80, 6982 (1983). 3. G. S. Wand, V. May, and B. A. Eipper, Endocrinology 123, 1153 (1988). 4. H. Shiomi, S. J. Watson, J. E. Kelsey, and H. Akil, Endocrinology 119, 1793 (1986). 5. J. H. Eberwine, C. Spencer, D. Newell, and A. R. Hoffman, Microsc. Res. Tech. 25, 19 (1993).
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19. 20. 21. 22. 23.
M. Kozak, J. Biol. Chem. 266, 19867 (1991). J. E. G. McCarthy and C. Gualerzi, Trends Genet. 6, 78 (1990). L. Gold, Annu. Rev. Biochem. 57, 199 (1988). M. Kozak, Annu. Rev. Biochem. 8, 197 (1992). R. D. Klausner, T. A. Rouault, and J. B. Harford, Cell 72, 19 (1993). C. M. Spencer and J. H. Eberwine, submitted (1994). J. D. Dignam, R. M. Lebovitz, and R. G. Roeder, Nucleic Acids Res. 11, 1475 (1983). L. P. Ovchinnikov, A. S. Spirin, B. Erni, and T. Staehelin, FEBS Left. 88, 21 (1978). T. Hunter, T. Hunt, R. J. Jackson, and H. D. Robertson, J. Biol. Chem. 250, 409 (1975). P. J. Farrell, G. C. Sen, M. E Dubois, L. Ratner, E. Slattery, and P. Lengyel, Proc. Natl. Acad. Sci. U.S.A. 75, 5893 (1978). E. A. Leibold and H. N. Munro, Proc. Natl. Acad. Sci. U.S.A. 85, 2171 (1988). M. M. Konarska and P. A. Sharp, Cell 46, 845 (1986). S. Pifiol-Roma, S. A. Adam, Y. D. Choi, and G. Dreyfuss, in "Methods in Enzymology," Vol. 180, p. 410. Academic Press, San Diego, California, 1989. U. K. Laemmli, Nature (London) 227, 680 (1970). A. Gatignol, A. Buckler-White, B. Berkhout, and K.- T. Jeang, Science 251, 1597 (1991). A. Gatignol and K.-T. Jeang, this series, Vol. 4, p. 18. T. A. Rouault, M. W. Hentze, D. J. Haile, J. B. Harford, and R. D. Klausner, Proc. Natl. Acad. Sci. US.A. 86, 5768 (1989). B. Neupert, N. A. Thompson, C. Meyer, and L. C. Kuhn, Nucleic Acids Res. 18, 51 (1990).
In Vitro Assays for Characterization
of RNA-Protein Complexes Involved in Pro-opiomelanocortin mRNA Translation Stephanie D. Flagg, Corinne M. Spencer, and James H. Eberwine
Introduction Proper regulation of pro-opiomelanocortin (POMC) gene expression is critical to the mammalian stress response. In response to physical or psychological stress, the hypothalamus secretes vasopressin and corticotropin-releasing hormone (CRH), which in turn stimulate POMC gene expression in the anterior and intermediate lobes of the pituitary. This stimulation results in the increased synthesis of POMC-derived peptides including adrenocorticotropin (ACTH) and/3-endorphin. Adrenocorticotropin then stimulates cortisol synthesis and secretion in the adrenal cortex. Glucocorticoids negatively regulate POMC gene expression in the anterior pituitary, while dopamine assumes this role in the intermediate lobe. Several groups have noted a discordance in the time courses over which POMC peptide and mRNA levels are altered in response to alterations in stress hormones ( 1 - 3). In pulse-chase experiments, Shiomi et al. (4) have noted a 50% increase in the efficiency of POMC peptide synthesis in rats exposed to acute stress at a time when no change in POMC mRNA levels is detected. Additionally, we have observed a change in the polysome distribution of POMC mRNA in response to treatment of AtT-2.0 cells (a mouse pituitary tumor) with dexamethasone or forskolin, a compound that mimicks the action of CRH (5). These findings suggest that POMC gene expression is regulated at the level of translation. Translational control refers to the regulation of any of several factors that determine the efficiency of protein synthesis. These factors include the rate at which ribosomal binding and initiation of translation occur, the rate at which each ribosome translocates along a mRNA molecule, and the fraction of mRNA that is actively being translated in polysomes. In addition, several features of a mRNA transcript are thought to influence its translation, including the exposure of the 7-methylguanosine cap, the context of the initiator methionine, and the degree of secondary and tertiary structure present within the mRNA (6). Control of gene expression at the level of translation allows for protein levels to be rapidly altered without necessitating a change in steady state mRNA transcript levels, thereby bypassing the time delay that is associated with RNA transcription, processing, and transport. The added fact that translation is an amplification process in which one mRNA molecule can be translated into thousands of molecules of proMethods in Molecular Genetics, Volume 8
Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
321
322
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TRANSCRIPTION
tein product allows this rapid control mechanism to alter protein levels dramatically. Translational control is thus a mechanism by which cells may rapidly respond to changes in the environment. One commonly utilized mechanism by which translational control is effected is through the interaction of an RNA-binding protein with a specific RNA sequence or structure. The binding of such proteins can alter the stability of RNA structures formed by intramolecular base pairing, which in turn can influence the efficiency by which a mRNA is translated. Protein-RNA interactions have been shown to impede the translation of many prokaryotic genes (7, 8), and have been demonstrated to play an important role in the expression of several eukaryotic genes as well (9, 10). Such protein-RNA interactions may play a role in the translational regulation of POMC gene expression. Computer modeling of POMC mRNA predicts the presence of a stem-loop structure of - 4 5 kcal/mol stability within the 5' coding region (Fig. 1). Data from in situ transcription experiments suggest that this predicted stemloop sequence exists in situ in rat pituitary tissue (5). On the basis of these data, we hypothesized that cytoplasmic proteins in rat pituitary and AtT-20 cells might recognize and bind to the stem-loop structure in POMC mRNA to effect its translational control. With the use of in vitro assays we have demonstrated the plausibility of this hypothesis. Cytoplasmic extracts from rat pituitary and AtT-20, but not COS cells (a monkey kidney fibroblast cell line), impede the translation of POMC mRNA in an in vitro translation assay (11). We have characterized RNA-protein interactions that play a role in POMC translation with the use of RNA gel mobility shift and ultraviolet (UV) cross-linking assays. These techniques are the focus of this chapter and are useful tools in the study of RNA-protein interactions that assume roles in RNA translation as well as those involved in RNA processing or transport.
In Vitro T r a n s c r i p t i o n
DNA sequences encoding the RNA sequence of interest should be cloned into a vector downstream of a strong bacteriophage RNA polymerase promoter such as T7, T3, or SP6. To obtain capped, full-length POMC mRNA that can be readily translated in rabbit reticulocyte lysates, we have used linearized DNA template and T7 RNA polymerase with the protocol for the mMessage mMachine in vitro transcription kit (Ambion, Inc.). For the production of large quantities of nonradioactive competitor RNAs, we have achieved optimal success with the use of the appropriate (T7, T3, or SP6) MEGAscript kit using the unmodified procedure as outlined by the manufacturer (Ambion, Inc.) or by using a high concentration of T7 RNA polymerase (1000 U//zl; Epicentre Technologies) as described below: 1. DNA template must be free of RNases and thus should be purified by cesium chloride gradient centrifugation rather than by commercially available column pro-
[17]
PROTEINS INVOLVED IN POMC mRNA TRANSLATION
AC C G C G A A C-G U-A C- G~-'~-- + 120 C "A A A G-C G-C A-U C-G C
323
(RAT=A)
Gu i G-C A-U C-G C-G G-C A-U
U C-G G-C A A G-C G-C UoG C-G C-G G-C
5'
3'
FIG. 1 Computer-predicted secondary structure of mouse POMC RNA stem-loop. The rat POMC stem-loop is identical to that of the mouse, with the exception of a single base pair substitution (A for G at position 120).
cedures, which rely on RNase treatment to remove contaminating RNA. DNA template should be linearized with the restriction enzyme that cuts 3' to the insert so as to minimize the amount of vector sequences transcribed and to ensure that all RNA transcripts synthesized will be of the same length. Restriction enzyme-cut plasmid DNA is extracted with equal volumes of phenol-chloroform and precipitated with a • vol of 3 M sodium acetate, pH 5.2, and 2.5 vol of ethanol. The pellet should be resuspended in Tris-EDTA (TE) buffer that has been filter sterilized. Because they stick to the nitrocellulose membrane, RNases can be removed from solutions during the filtration process. 2. Set up in vitro transcription reactions in RNase-free microfuge tubes. All buff-
324
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ers are made RNase free by sterile filtration or by treatment of buffers that do not contain primary amines with diethyl pyrocarbonate (DEPC). H20 Transcription buffer (5 • 200 mM Tris (pH 7.5), 35 mM MgC12, 50 mM NaC1, 10 mM spermidine Dithiothreitol (DTT, 100 mM) RNasin (40 U; Promega, Madison, WI) ATP (10 mM) CTP (10 mM) GTP (10 mM) UTP (10 mM) Linearized template DNA T7 RNA polymerase (1000 U; Epicentre Technologies)
43/zl 20/zl 10/xl
1/xl 5 #l 5/xl 5/xl 5/zl 5/xl = 1/xg
1/xl
3. Incubate the reactions for 90 min at 37 ~C. 4. Remove the DNA template by addition of 2/zl of RNase-free DNase I (10 U/ /zl; BMB) and continue the incubation at 37~ for 30 min. 5. Extract the RNA with phenol-chloroform, and ethanol precipitate. 6. Resuspend the RNA in 10 #1 of sterile-filtered H20 and drop dialyze the sample on nitrocellulose filters (Cat. No. VSWP01300; Millipore, Bedford, MA) for 1-4 hr to remove free triphosphates. Repeat the phenol-chloroform extraction and ethanol precipitation. 7. Obtain a spectrophotometric reading of sample at 260 nm to quantitate the RNA. Radiolabeled riboprobe can be synthesized by the above protocol if 30/xCi of [ce-32p]CTP is added to the reaction tubes and the final concentration of unlabeled CTP is reduced to 0.5/xM. T7 RNA polymerase of a lower specific activity (GIBCOBRL, Gaithersburg, MD) may be employed in riboprobe synthesis if a smaller quantity of RNA needs to be produced. Additionally, free triphosphates can be removed with two sequential ethanol precipitations. Counts per minute are determined by scintillation counting.
Cytoplasmic Extract Preparation S 100 Cytoplasmic extracts are prepared as described (12). Briefly: 1. Grow cells in culture to 5 • 105 and harvest by centrifugation for 10 min at 2000 rpm in a Sorvall HG4L rotor. All subsequent steps should be performed at 4 ~C. 2. Resuspend the pellet in 5 vol of phosphate-buffered saline.
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3. Centrifuge as in step 1. 4. Resuspend in five packed cell pellet volumes of buffer A [10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES, pH 7.9 at 4~ 1.5 mM MgCI2, 10 mM KC1, 0.5 mM DTT] and incubate for 10 min on ice. 5. Centrifuge as in step 1. 6. Resuspend the pellet in 2 vol of buffer A, and lyse cells with at least 10 strokes of an all-glass Kontes Dunce homogenizer, type B pestle. The homogenate should be checked at this point for the efficiency of cell lysis. For effective lysis of AtT-20 cells, an additional one or two rounds of homogenization are required. 7. Centrifuge as in step 1 to pellet nuclei. 8. Mix the resulting supernatant with 0.11 vol of buffer B [300 mM HEPES (pH 7.9 at 4~ 1.4 M KC1, 30 mM MgC12] and centrifuge for 60 min at 100,000 gav. 9. Dialyze the supernatant for 5 to 8 hr against 20 vol of buffer D [20 mM HEPES (pH 7.9 at 4~ 100 mM KC1, 0.2 mM EDTA, 20% (v/v) glycerol, 0.1 M KC1, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5 mM DTT]. Protein determinations can be made by the use of the Bradford assay (Bio-Rad, Richmond, CA). Aliquot cytoplasmic extracts and store at - 8 0 ~C. Note: Dithiothreitol and PMSF should be added to buffers at the time of use.
In Vitro T r a n s l a t i o n s
We have used a rabbit reticulocyte lysate system (Amersham, Arlington Heights, IL) to demonstrate the ability of protein-RNA interactions to regulate the in vitro translation of POMC mRNA (Fig. 2). Reaction conditions are outlined by the manufacturer. Briefly, a 10-/zl reaction is set up consisting of 200 ng of the full-length mRNA of interest (or control RNA such as brome mosaic virus; Amersham), 10 #Ci of L-[35S]methionine (translation grade; Amersham), 4 /zl of cytoplasmic extract or Dignam buffer D (see cytoplasmic extract preparation), and 4 #1 of nuclease-treated, message-dependent rabbit reticulocyte lysate. Translation reactions are incubated for 1 hr (time course should be empirically determined) at 30~ and then mixed with 2• sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, boiled for 5 min, and electrophoresed on the percentage denaturing polyacrylamide gels that will achieve effective separation of the translated protein product. After treatment with Entensify universal autoradiography enhancer (NEN-Dupont, Boston, MA), gels are dried and exposed to film for 3 - 7 days at -80~ Competitor RNAs can be included in the translation reactions to evaluate the ability of an RNA sequence or structure to compete with the full-length mRNA for interaction with cytoplasmic protein and attenuate the ability of cytoplasmic extract to alter the translation of the full-length mRNA. Certain issues must be kept in mind in performing these experiments and in interpreting the resulting data. High concentrations of RNA inhibit protein synthesis in rabbit reticulocyte lysates, perhaps by sequestering initia-
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lxg AtT20 Extract
6.4
0
1.6
3.2
200 ng POMC RNA
-
+
+
+ -- 221.2 kDa
-- 105.7 kDa
-- 74.7 kDa
-- 46.3 kDa
"- 27.6 kDa
"- 18.6 kDa
FIG. 2 The translation of P O M C RNA in vitro is inhibited by AtT-20 cytoplasmic extract. Two hundred nanograms of capped full-length P O M C m R N A was translated in a rabbit reticulocyte lysate system in the presence of increasing amounts of AtT-20 cytoplasmic extract (lanes 2 - 4 ) . The first lane is a control reaction in which no RNA was added to the reticulocyte lysate. The arrow indicates the position of the P O M C peptide as determined by immune precipitation (data not shown).
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tion and elongation factors (13). Furthermore, the addition of high concentrations (> 1 ~g/ml) of double-stranded RNA to reticulocyte lysate can lead to a generalized translational arrest (14, 15) by blocking the activation of a double stranded RNAdependent protein kinase that phosphorylates initiation factor eIF-2. Thus, proper controls are required to evaluate data from these experiments.
R N A Gel M o b i l i t y Shift A s s a y RNA gel mobility shift assays are performed essentially as described (16). 1. Set up RNA-protein binding reactions in RNase-free microfuge tubes with 5 25 #g of cytoplasmic extracts (or lesser amounts of purified protein), 40 U of RNasin (Promega), and 50,000 cpm of labeled riboprobe in 10 mM HEPES (pH 7.6), 3 mM MgCI2, 40 mM KC1, 5% (v/v) glycerol, and 1 mM DTT in a 20-/.~1 reaction volume. One reaction should be set up with riboprobe in the absence of protein extract to demonstrate the mobility of unretarded RNA transcript. 2. Incubate binding reactions at room temperature for 20 rain to allow proteins to interact with the riboprobe. Salt concentrations, incubation time, and temperature may need to be modified to optimize binding. . 3. Add 1 #1 of heparin (100 mg/ml; Sigma, St. Louis, MO) and continue the incubation at room temperature for 10 min. The addition of heparin should decrease the binding of proteins that interact nonspecifically with the phosphate backbone of the riboprobe. 4. Electrophoresis is performed as described (Ref. 17, with modifications). Load binding reactions directly onto a 5% (w/v) nondenaturing polyacrylamide (60: 1, acrylamide-bisacrylamide) gel that has been preelectrophoresed for 30-60 min at 8 V/cm in 45 mM Tris-borate, 1 mM EDTA. Electrophorese the gel for 2 to 2.5 hr at 8 V/cm. 5. Dry the gel and autoradiograph at room temperature for 12 to 24 hr. The RNA gel mobility shift assay is a rapid method that can be used to demonstrate whether a purified protein or proteins within an extract preparation can bind specifically to an RNA fragment. Control reactions performed in the absence of protein demonstrate the unretarded migration of riboprobe within the gel. The addition of cytoplasmic extract containing proteins that recognize the riboprobe results in the formation of riboprobe-protein complexes that are distinguishable by their slower migration through the gel (Fig. 3). Competition experiments can be performed by the inclusion of a 10- to 1000-fold molar excess of unlabeled competitor RNAs to the binding reactions (step 1). Unlabeled competitor RNAs that specifically compete with the riboprobe for protein binding will disrupt the formation of riboprobe-protein complexes (less riboprobe
328
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TRANSCRIPTION
AtT20 Extract
-
+
+
Sense Competitor RNA
.
.
+
Complex 2 Complex 1
Free Riboprobe
FIG. 3 RNA gel mobility shift assay with a 32p-labeled POMC stem-loop RNA in the absence (lane 1) or presence (lane 2) of 5/zg of cytoplasmic AtT-20 extract. Two RNA protein complexes are observed on the addition of AtT-20 cytoplasmic extract. These complexes are competed away by the addition of 100 ng of unlabeled POMC stem-loop RNA (lane 3).
will be shifted) to a greater degree than will cold nonspecific RNAs. Nonspecific RNAs that are commonly used in this assay include RNAs of the same length as the riboprobe that are synthesized from linearized vector DNA templates, tRNA, and poly(rI-rC). Proteinase K should be included in a control reaction to demonstrate unequivocally that the retarded migration of riboprobe is due to the interaction of protein with the RNA. The RNA gel mobility shift assay does not provide information on the molecular weight of the proteins that interact with the riboprobe, and does not indicate the number of proteins that contribute to the riboprobe-protein complexes formed. For this type of information, a UV cross-linking assay may be performed.
Ultraviolet Cross-Linking Assay Binding reactions are performed as described in steps 1 to 3 for the gel mobility shift assay, except that reactions are set up in the caps of microfuge tubes. Competition experiments can be performed by adding unlabeled competitor RNAs in the binding reactions as was described for the gel mobility shift assay. After the addition of heparin:
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1. Binding reactions are exposed to UV light (Sylvania G15T8; GTE Sylvania, Inc.) for 10 min at a distance of 4.5 cm (18). A time course should be performed with the UV source at various distances to optimize conditions. 2. Incubate the reactions at room temperature with 5 /xg of RNase A to digest RNA not protected by covalent linkage to protein. Some protocols include a variety of RNases (T1, T2, and V1) in this incubation. RNase A cleaves after pyrimidine residues; RNase T1 cuts after guanosine residues; T2 cleaves after any residue; and RNase VI cleaves double-stranded RNA. The addition of these enzymes to the reactions may be important for the complete digestion of highly structured RNAs. 3. Add an equal volume of 2• SDS sample buffer to the reaction tubes, and boil the samples for 5 min before separating the proteins by electrophoresis on 8-10% (w/v) SDS-polyacrylamide gels according to a standard protocol (19). 4. Dry the gel and visualize complexes by autoradiography. Ultraviolet treatment of the binding reactions covalently cross-links to RNA proteins that are in direct association with it. RNase treatment of the RNA-protein complexes degrades all riboprobe that is not protected by direct cross-links to protein. When these samples are run on denaturing polyacrylamide gels, the proteins that are cross-linked to small fragments of riboprobe are separated by virtue of their molecular weight and can be visualized by autoradiography. This assay allows for the estimation of molecular weight of the RNA-binding proteins and gives information as to the number of proteins that can bind directly to the riboprobe (Fig. 4). This technique, however, has several limitations. A single band on a UV cross-linking experiment does not correspond necessarily to one unique RNA-binding protein because one band may represent either a complex of proteins (if two or more proteins become cross-linked by the UV treatment) or the partial degradation product of an RNA-binding protein. In addition, a protein that binds indirectly to the RNA via a protein-protein interaction may not become covalently cross-linked, and therefore would not be visualized on autoradiography.
In Vitro M u t a g e n e s i s o f R N A R e c o g n i t i o n Site The RNA-binding site recognized by extract proteins can be further delineated by site-directed mutagenesis of the RNA fragment. Mutant RNAs can be synthesized from mutagenized DNA templates and used as riboprobes or competitors in gel mobility shift and UV cross-linking assays. Additionally, mutant RNAs can be studied in in vitro translation assays and in vivo, after transfection of cells with reporter gene constructs. From these types of experiments, one can ascertain the degree to which sequence and structure of an RNA contribute to the RNA-protein interactions being studied. We have created mutants in the stem-loop structure of POMC mRNA with the use
330
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min UV
0
10
30
--221 kDa
-- 106 k D a
- 75 k D a
-- 4 6 k D a
FIG. 4 Ultraviolet cross-linking experiment with 32p-labeled POMC RNA stem-loop and 18.7 /xg of AtT-20 cytoplasmic extracts. Samples were exposed to UV light for 0, 10, or 30 minutes as indicated (lanes 1-3). Four RNA-protein complexes are observed.
of the Altered Sites in vitro mutagenesis system (Promega). This system requires subcloning the DNA encoding the RNA of interest into the multiple cloning site of the pSELECT-1 phagemid-plasmid, which contains SP6 and T7 promoters. Singlestranded DNA (ssDNA) is produced by infection of host bacterial cells with helper phage. This ssDNA is annealed to a 5'-phosphorylated mutant oligonucleotide and the synthesis of the mutant strand is completed with the addition of T4 DNA polymerase and T4 DNA ligase. The percentage of mutants that is created is optimized by performing a first round of transformation in BMH 71-18 MutS, a repair-defective Escherichia coli strain. A second round of transformation is performed in JM109 cells in order to ensure segregation of wild-type and mutant clones. Clones are sequenced to confirm the identity of mutants. The pSELECT-1 plasmid contains a gene for tetracycline resistance and a nonfunctional ampicillin resistance gene. This feature, and the provision of an oligonucleotide that when included in the annealing reaction results in the repair of the ampicillin resistance gene, allows for
[17] PROTEINSINVOLVEDIN POMC mRNATRANSLATION
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ampicillin selection for mutant strand synthesis and increases the percentages of mutants obtained. A newer version of this system (Altered Sites II in vitro mutagenesis system; Promega) is currently available that boasts as advantages (a) the inclusion of a protocol for mutagenesis of double-stranded DNA and (b) the ability to perform multiple rounds of mutagenesis without resubcloning by alternating the knockout and repair of the tetracycline and ampicillin genes. We have found the ES 1301 MutS repairdefective E. coli strain that currently must be used for multiple rounds of mutagenesis to be difficult to transform. Competent ES 1301 MutS cells soon may be available from Promega.
Summary Although we have chosen to illustrate the utility of these techniques in the characterization of RNA-binding proteins that are involved in translation, these techniques are also useful in characterizing RNA-binding proteins that have other functional roles within the cell, such as the processing and transport of RNA. In particular, these techniques are being utilized to characterize RNA-binding proteins involved in the subcellular distribution of RNAs as well as those thought to be involved in regulating the course of various neurological diseases. Once the specificity of an RNA-protein interaction has been characterized by these methods, one can isolate the protein(s) involved by the use of a direct cloning approach via a Northwestern hybridization screen (20, 21) or by biochemical purification techniques that take advantage of RNA affinity chromatography (22, 23).
Acknowledgments J.H.E. is an established investigator of the American Heart Association. S.D.F. is supported by NIGMS and is a Dupont-Merck Scholar.
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19. 20. 21. 22. 23.
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