Reagents and methods for identification of RNAi pathway genes and chemical modulators of RNAi

• US 20050266552A1
• Filed: 12/05/2004
• Published: 12/01/2005
• Est. Priority Date: 12/05/2003
• Status: Abandoned Application

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Novina, Carl D., Doench, John G., Dykxhoorn, Derek M., Sharp, Phillip A.
• Application Number:
  US11/005,127
• Publication Number:
  US 20050266552A1
• Time in Patent Office: Days
• US Class Current: 435/358
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2320/12 in functional genomics, i.e. for the determination of gene function
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/5041 involving analysis of members of signalling pathways

Abstract

The present invention provides reagents such as cells, cell lines, and vectors, that can be used to identify mammalian genes whose expression products (RNA or protein) play a role in RNA interference (RNAi) and/or to identify chemical modulators of RNAi, or for other purposes. The invention further provides a variety of methods for identifying such genes or modulators. In particular, the invention provides a mammalian cell comprising a nucleic acid that encodes a selectable marker and one or more nucleic acid templates for transcription of an RNAi-inducing agent integrated into the genome of the cell, wherein the RNAi-inducing agent reduces expression of the marker and is not naturally found in the cell. Additional cells and cell lines comprising nucleic acids that encode one or more additional markers are also provided. According to certain of the inventive methods cells such as these are mutagenized, transfected or infected with a library of genetic suppressor elements, or contacted with a test compound. Cells in which RNAi is inhibited or activated are identified using an appropriate selective condition or screening method. The identity of the mutated or inhibited gene or the identity of the compound is then determined.

Claims

1. A mammalian cell comprising:

(i) a nucleic acid that encodes a selectable marker; and

(ii) one or more nucleic acid templates for transcription of an RNAi-inducing agent integrated into the genome of the cell, wherein the RNAi-inducing agent reduces expression of the marker and is not naturally found in the cell.

2. The cell of claim 1, wherein the cell is a human cell.

3. The cell of claim 1, wherein the cell is a HeLa cell.

4. The cell of claim 1, wherein the cell is a non-human cell.

5. The cell of claim 1, wherein the cell is hypodiploid.

6. The cell of claim 1, wherein the cell is a CHO cell.

7. The cell of claim 1, wherein the selectable marker is an expression product of an endogenous gene.

8. The cell of claim 1, wherein the nucleic acid that encodes a selectable marker is operably linked to an inducible promoter.

9. The cell of claim 1, wherein the nucleic acid that encodes a selectable marker is operably linked to a constitutive promoter.

10. The cell of claim 1, wherein the cell further comprises:

(i) a nucleic acid that encodes a detectable marker; and

(ii) one or more templates for transcription of an RNAi-inducing agent that reduces expression of the detectable marker.

11. The cell of claim 10, wherein a template for transcription of the RNAi-inducing agent that reduces expression of the detectable marker is integrated into the genome of the cell.

12. The cell of claim 1, 10, or 11, wherein the RNAi-inducing agent is a short hairpin RNA (shRNA).

13. The cell of claim 1, 10, or 11, wherein the RNAi-inducing agent is an siRNA or precursor thereof.

14. The cell of claim 1, 10, or 11, wherein the RNAi inducing agent is a precursor of a microRNA (miRNA).

15. The cell of claim 1, 10, or 11, wherein the cell further comprises:

(i) a nucleic acid that encodes a second selectable marker; and

(ii) one or more templates for transcription of an RNAi-inducing agent that reduces expression of the second selectable marker.

16. The cell of claim 10 or 15, wherein the detectable marker produces a fluorescent, luminescent, or colorimetric-signal.

17. The cell of claim 16, wherein the detectable marker is selected from the group consisting of:

green fluorescent protein, an enhanced green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, cyan fluorescent protein.

18. The cell of claim 16, wherein the detectable marker comprises a region that targets the marker for intracellular proteolysis.

19. The cell of claim 18, wherein the region is a PEST domain.

20. The cell of claim 18, wherein the detectable marker is a fusion protein having the region that targets the marker for increased intracellular proteolysis located at its N-terminus or C-terminus.

21. The cell of claim 16, wherein the detectable marker has a half-life of approximately 2 hours or less.

22. The cell of claim 16, wherein the detectable marker has a half-life of approximately 1 hour or less.

23. The cell of claim 16, wherein the detectable marker is enhanced GFP having increased expression level, increased fluorescence intensity, decreased half-life, or a combination of any of the foregoing relative to wild type GFP.

24. The cell of claim 23, wherein the enhanced GFP has a decreased half-life relative to wild type GFP.

25. The cell of claim 1, wherein the nucleic acid that encodes a selectable marker is integrated into the genome of the cell.

26. The cell of claim 1, wherein expression of the selectable marker confers a growth advantage under a first set of selective conditions and confers a growth disadvantage under a second set of selective conditions.

27. The cell of claim 1, wherein the selectable marker is selected from the group consisting of:

proteins that catalyze a step in a nucleotide synthesis or salvage pathway, DNA repair proteins, DNA synthesis proteins, RNA synthesis proteins, proteins that expel a cytotoxic compound out of a cell, and proteins that alter the permeability of a cell to a cytotoxic compound.

28. The cell of claim 1, wherein the selectable marker is selected from the group consisting of:

HPRT, TK, DHFR, and MDR family members.

29. The cell of claim 1, wherein the selectable marker is HPRT.

30. The cell of claim 1, wherein the selectable marker confers resistance to an antibiotic.

31. The cell of claim 1, wherein the selectable marker confers resistance to a chemotherapeutic agent.

32. The cell of claim 31, wherein the chemotherapeutic agent is selected from the group consisting of:

methotrexate, vinblastine, and anthracyclines.

33. The cell of claim 1, further comprising:

(i) a nucleic acid that encodes a second marker, wherein the marker is a selectable or detectable marker;

(ii) one or more nucleic acid templates for transcription of an RNAi-inducing agent, wherein the RNAi-inducing agent reduces expression of the second marker, and wherein the templates for transcription of RNAi-inducing agents that reduce expression of the markers are integrated into the genome of the cell as a single unit.

40. A cell line comprising a plurality of cells as set forth in claim 1, 2, 5, 10, 15, 26, 33, 34, or 39, wherein the cells are descended from a single cell.

52. A kit comprising the collection of cell lines of claim 41 and one or more items selected from the group consisting of:

(i) an RNAi-inducing agent that targets an mRNA that encodes the marker;

(ii) an RNAi-inducing agent that does not target an mRNA that encodes the marker;

(iii) a compound that inhibits RNAi;

(iv) a compound that activates RNAi;

(v) a genetic element that inhibits RNAi;

(vi) a genetic element that activates RNAi;

(vii) an RNAi-inducing agent that targets an mRNA that encodes Dicer;

(viii) a cell line comprising a plurality of the mammalian cell of claim 1;

(ix) one or more compounds for addition to tissue culture medium to impose a selective condition on the mammalian cell line of (viii);

(x) a cell line that comprises a nucleic acid that encodes the same marker as the collection of cell lines but does not comprise a template for transcription of an RNAi-inducing agent that reduces expression of the marker;

(xi) a vector comprising a U6, H1, or tRNA promoter and a site downstream of the promoter for insertion of a template for transcription of an RNAi-inducing agent;

(xii) a transfection reagent; and

(xiii) instructions for use.

53. The kit of claim 52, wherein the marker of the collection of cell lines is a detectable marker.

54. A kit comprising a cell line comprising a plurality of cells as set forth in claim 1 and one or more items selected from the group consisting of:

(i) an RNAi-inducing agent that targets an mRNA that encodes the selectable marker;

(ii) an RNAi-inducing agent that does not target an mRNA that encodes the selectable marker;

(iii) a cell line that comprises a nucleic acid that encodes the same marker as the cell line comprising a plurality of cells as set forth in claim 1 but does not comprise a template for transcription of an RNAi-inducing agent that reduces expression of the marker;

(iv) one or more of the cell lines of the collection of cell lines of claim 41;

(v) an RNAi-inducing agent that targets an mRNA that encodes the marker of the cell line of (iv);

(vi) a compound that inhibits RNAi;

(vii) a compound that activates RNAi;

(viii) a genetic element that inhibits RNAi;

(ix) a genetic element that activates RNAi;

(x) an RNAi-inducing agent that targets an mRNA that encodes Dicer;

(xi) one or more compounds for addition to tissue culture medium to impose a selective condition on the cell line comprising a plurality of the cell of claim 1;

(xii) a cell line that comprises a nucleic acid that encodes the same marker as the collection of cell lines but does not comprise a template for transcription of an RNAi-inducing agent that reduces expression of the marker;

(xiii) a vector comprising a U6, H1, or tRNA promoter and a site downstream of the promoter for insertion of a template for transcription of an RNAi-inducing agent; and

(xiv) instructions for use.

55. The kit of claim 54, wherein the marker of the collection of cell lines is a detectable marker.

34. A cell comprising:

(i) a nucleic acid that encodes a detectable marker, wherein the detectable marker has a half-life of approximately 2 hours or less; and

(ii) a template for transcription of an RNAi-inducing agent that reduces expression of the detectable marker integrated into the genome of the cell, wherein the RNAi-inducing agent reduces expression of the marker and is not naturally found in the cell.

35. The cell of claim 34, wherein the detectable marker has a half-life of approximately 1 hour or less.

36. The cell of claim 34, wherein the marker is enhanced GFP having increased expression level, increased fluorescence intensity, decreased half-life, or a combination of any of the foregoing relative to wild type GFP.

37. The cell of claim 36, wherein the enhanced GFP has a decreased half-life relative to wild type GFP.

38. A mammalian cell comprising:

a nucleic acid that is integrated into the genome of the cell and provides a template for transcription of an mRNA transcript that encodes a detectable or selectable marker, wherein the mRNA transcript comprises one or more binding sites for an endogenous miRNA or an miRNA-like RNA; and

an endogenous miRNA or miRNA-like RNA that is expressed by the cell and represses translation of the mRNA that encodes a detectable or selectable marker.

39. The mammalian cell of claim 38, wherein the cell expresses an siRNA or shRNA that is targeted to the mRNA transcript.

41. A collection of cell lines wherein cells of each cell line comprise:

(i) a nucleic acid that encodes a marker, wherein the nucleic acid in cells of each cell line encodes the same marker; and

(ii) a template for transcription of an RNAi-inducing agent that reduces expression of the marker, wherein the RNAi-inducing agent reduces expression of the marker to different extents in each of the cell lines.

42. The collection of cell lines of claim 41, wherein each of the cell lines is derived from a single cell.

43. The collection of cell lines of claim 41, wherein the RNAi-inducing agent in each of the cell lines is the same.

44. The collection of cell lines of claim 41, wherein the cell lines comprise cells of the same cell type.

45. The collection of cell lines of claim 41, wherein the cell lines are human cell lines.

46. The collection of cell lines of claim 41, wherein the cell lines are non-human cell lines.

47. The collection of cell lines of claim 41, wherein the marker is a detectable marker.

48. The collection of cell lines of claim 41, wherein the detectable marker has a half-life of approximately an hour or less.

49. The collection of cell lines of claim 41, wherein the marker is enhanced GFP having increased expression level, increased fluorescence intensity, decreased half-life, or a combination of any of the foregoing relative to wild type GFP.

50. The collection of cell lines of claim 49, wherein the enhanced GFP has a decreased half-life relative to wild type GFP.

51. The collection of cell lines of claim 41, further comprising a cell line that comprises a nucleic acid that encodes the marker but does not comprise a template for transcription of an RNAi-inducing agent that reduces expression of the marker.

56. A cell comprising:

(i) a nucleic acid that encodes a marker, wherein expression of the marker increases or decreases sensitivity of the cell to a compound or environmental condition so that growth of the cell is inhibited or enhanced, respectively, in the presence of the compound or environmental condition relative to growth in its absence; and

(ii) one or more templates for transcription of an RNAi-inducing agent that reduces expression of the marker so that growth of the cell in the presence of the compound or environmental condition is increased or decreased relative to growth in its absence.

57. The cell of claim 56, wherein expression of the marker increases sensitivity of the cell to a compound or environmental condition so that growth of the cell is inhibited in the presence of the compound or environmental condition relative to growth in its absence, and wherein the RNAi-inducing agent reduces expression of the marker so that growth of the cell in the presence of the compound or environmental condition is increased relative to growth in its absence.

58. The cell of claim 56, wherein expression of the marker decreases sensitivity of the cell to a compound or environmental condition so that growth of the cell is enhanced in the presence of the compound or environmental condition relative to growth in its absence, and wherein the RNAi-inducing agent reduces expression of the marker so that growth of the cell in the presence of the compound or environmental condition is decreased relative to growth in its absence.

59. The cell of claim 56, wherein the marker is selected from the group consisting of:

HPRT, TK, DHFR, and MDR family members.

60. A nucleic acid comprising:

(i) a template for transcription of a first RNAi-inducing agent targeted to an mRNA that encodes a first marker, wherein the template is operably linked to a promoter active in a mammalian cell; and

(ii) a template for transcription of a second RNAi-inducing agent targeted to an mRNA that encodes a second marker, wherein the template is operably linked to a promoter active in a mammalian cell.

61. The nucleic acid of claim 60, wherein transcription from the promoters is driven by RNA polymerase I or RNA polymerase III.

62. The nucleic acid of claim 61, wherein the promoter is selected from the group consisting of:

the U6 promoter, the H1 promoter, and tRNA promoters.

63. The nucleic acid of claim 60, wherein transcription from at least one of the promoters is driven by RNA polymerase II.

64. The nucleic acid of claim 60, wherein one or both of the RNAi-inducing agents are shRNAs.

65. The nucleic acid of claim 60, wherein one or both of the RNAi-inducing agents are siRNAs or precursors thereof.

66. The nucleic acid of claim 60, wherein one or both of the RNAi-inducing agents are precursors of miRNAs.

67. The nucleic acid of claim 60, wherein one or both of the markers is a selectable marker.

68. The nucleic acid of claim 60, wherein one or both of the markers is a detectable marker.

69. The nucleic acid of claim 60, wherein one of the markers is a detectable marker and the other marker is a selectable marker.

70. A vector comprising the nucleic acid of claim 60.

71. A mammalian cell comprising the nucleic acid of claim 60.

72. A cell line comprising a plurality of cells as set forth in claim 71.

73. A nucleic acid comprising (i) a template for transcription of a first RNAi-inducing agent targeted to a selectable or detectable marker and operably linked to a first promoter and (ii) a second promoter and a site for insertion of a template for transcription of an RNAi-inducing agent located downstream of the promoter, so that the template will be operably linked to the promoter once inserted.

74. The nucleic acid of claim 73, further comprising a region that encodes the selectable or detectable marker.

75. The nucleic acid of claim 73, wherein the marker is a selectable marker selected from the group consisting of HPRT, TK, or an MDR family member.

76. A vector comprising the nucleic acid of claim 73.

77. A mammalian cell comprising the nucleic acid of claim 73.

78. A cell line comprising a plurality of cells as set forth in claim 77.

79. A method of identifying a cell in which a gene of interest is silenced by RNAi comprising steps of:

(i) introducing the nucleic acid of claim 73 into a population of cells, wherein the nucleic acid further comprises a template for transcription of an RNAi-inducing agent targeted to the gene of interest; and

(ii) identifying a cell in which RNAi is active by selecting or detecting cells that do not express the selectable or detectable marker, thereby identifying a cell in which the gene of interest is silenced by RNAi.

80. The method of claim 79, wherein the marker is a selectable marker and the step of identifying comprises exposing the cells to selective conditions that select against cells that express the selectable marker.

81. The method of claim 79, wherein the marker is an endogenous gene.

82. The method of claim 79, wherein the marker is selected from the group consisting of HPRT, TK, or an MDR family member.

83. The method of claim 79, wherein the step of identifying comprises exposing the cells to a compound that is processed by the selectable marker to yield a toxic compound.

84. The method of claim 79, wherein the marker is a detectable marker and the step of identifying comprises detecting a cell that does not express the marker.

85. A method of identifying a gene involved in an RNAi pathway comprising steps of:

(a) providing a population of mammalian cells members of which comprise a nucleic acid that encodes a detectable or selectable marker and further comprise one or more templates for transcription of an RNAi-inducing agent that reduces expression of the detectable or selectable marker;

(b) mutagenizing the population of cells; and

(c) identifying cells that display decreased or increased expression of the detectable or selectable marker relative to the starting population, thereby identifying cells that have a mutation in a gene involved in an RNAi pathway.

86. The method of claim 85, wherein the identifying step comprises identifying cells that display decreased expression of the marker relative to the starting population, thereby identifying cells that have a gain of function mutation in a gene involved in an RNAi pathway.

87. The method of claim 85, wherein the identifying step comprises identifying cells that display increased expression of the marker relative to the starting population, thereby identifying cells that have a loss of function mutation in a gene involved in an RNAi pathway.

88. The method of claim 85, wherein the marker is a selectable marker.

89. The method of claim 88, wherein the marker is selected from the group consisting of:

HPRT, TK, DHFR, and MDR family proteins.

90. The method of claim 85, wherein the marker is a detectable marker.

91. The method of claim 90, wherein the detectable marker comprises a domain that increases intracellular proteolysis of the marker.

92. The method of claim 85, wherein the cell comprises a nucleic acid that encodes a detectable marker and a nucleic acid that encodes a selectable marker.

93. The method of claim 85, wherein the step of mutagenizing is performed by exposing the cells to a chemical mutagen or to radiation.

94. The method of claim 85, wherein the step of mutagenizing comprises insertional mutagenesis.

95. The method of claim 94, wherein insertional mutagenesis is performed by infecting the cells with a retrovirus.

96. The method of claim 95, further comprising the step of recovering the virus.

97. The method of claim 85, further comprising the step of:

performing a secondary screen or selection wherein the secondary screen or selection assesses the ability of an RNAi-inducing agent targeted to a second marker to inhibit expression of the second marker in the cell.

98. The method of claim 97, wherein the second marker is a detectable marker.

99. The method of claim 97, wherein the second marker is a selectable marker.

100. The method of claim 85, further comprising the step of:

cloning the gene having the mutation.

101. A method of identifying a gene involved in a miRNA translational repression pathway comprising steps of:

(a) providing a population of mammalian cells members of which comprise (i) a nucleic acid that is integrated into the genome of the cell and provides a template for transcription of an mRNA transcript that encodes a detectable or selectable marker, wherein the mRNA transcript comprises one or more binding sites for an endogenous miRNA or an miRNA-like RNA; and

(ii) an endogenous miRNA or miRNA-like RNA that is expressed by the cell and represses translation of the mRNA that encodes a detectable or selectable marker;

(b) mutagenizing the population of cells; and

(c) identifying cells that display decreased or increased expression of the detectable or selectable marker relative to the starting population and do not display an alteration in mRNA transcript level sufficient to account for the increased or decreased expression of the marker, thereby identifying cells that have a mutation in a gene involved in an miRNA translational repression pathway.

102. The method of claim 101, further comprising the step of:

cloning the gene.

154. A computer-readable medium containing computer-readable information indicating that a gene, mutation, genetic element, or compound affects an RNAi pathway, wherein the gene, mutation, genetic element, or compound was identified as affecting an RNAi pathway by the method of claim 85, 101, 103, 133, 135, or 153.

103. A method for identifying cells containing a genetic element that inhibits or activates an RNAi pathway comprising steps of:

(a) providing a first population of mammalian cells members of which comprise a nucleic acid that encodes a first detectable or selectable marker and express an RNAi-inducing agent that reduces expression of the detectable or selectable marker;

(b) introducing a library into the population of cells, wherein the library comprises a plurality of genetic elements;

(c) identifying cells that display increased or decreased expression of the detectable or selectable marker relative to the starting population, thereby identifying cells that contain a genetic element that inhibits or activates an RNAi pathway, respectively.

104. The method of claim 103, wherein the identifying step comprises identifying cells that display increased expression of the detectable marker relative to the starting population, thereby identifying cells that contain a genetic element that inhibits an RNAi pathway.

105. The method of claim 103, wherein the identifying step comprises identifying cells that display decreased expression of the detectable marker relative to the starting population, thereby identifying cells that contain a genetic element that activates an RNAi pathway.

106. The method of claim 103, wherein the genetic element is a genetic suppressor element.

107. The method of claim 103, wherein the marker is a selectable marker.

108. The method of claim 103, wherein the marker is a detectable marker.

109. The method of claim 103, further comprising the step of identifying the genetic element.

110. The method of claim 109, wherein the step of identifying the genetic element comprises PCR amplification.

111. The method of claim 109, wherein the step of identifying the genetic element comprises sequencing.

112. The method of claim 103 or 109, further comprising the step of identifying the gene suppressed by the genetic element, thereby identifying a gene involved in an RNAi pathway.

113. The method of claim 103, wherein the step of introducing the library comprises retroviral infection.

114. The method of claim 103, wherein the step of introducing the library comprises DNA transfection.

115. The method of claim 103, wherein the library is a cDNA library.

116. The method of claim 115, wherein the cDNAs are size-selected.

117. The method of claim 115, wherein the cDNAs are normalized.

118. The method of claim 115, wherein some or all of the cDNAs encode protein fragments.

119. The method of claim 115, wherein some or all of the cDNAs are operably linked in reverse orientiation to a promoter so that transcription results in synthesis of antisense RNA molecules.

120. The method of claim 103, wherein the templates for transcription are inserted into a vector comprising an episomal element that replicates within mammalian cells.

121. The method of claim 103, wherein the templates for transcription are inserted into a retroviral vector.

122. The method of claim 103, further comprising the step of:

performing a secondary screen or selection.

123. The method of claim 122, wherein the secondary screen or selection assesses the ability of an RNAi-inducing agent targeted to a second marker to inhibit expression of the second marker in the cell.

124. The method of claim 122, wherein the second marker is a detectable marker.

125. The method of claim 122, wherein the second marker is a selectable marker.

126. The method of claim 122, wherein the secondary screen or selection comprises:

(i) introducing the genetic element into a second population of mammalian cells members of which comprise a nucleic acid that encodes a second detectable or selectable marker and express an RNAi-inducing agent that reduces expression of the second detectable or selectable marker; and

(ii) assessing expression of the second detectable or selectable marker.

127. The method of claim 126, wherein the second marker is a detectable marker and the step of assessing expression of the marker comprises detecting the marker.

128. The method of claim 126, wherein the second marker is a selectable marker and the step of assessing expression of the marker comprises detecting cell growth under selective conditions.

129. A genetic element identified according to the method of claim 109.

130. A protein fragment encoded by a genetic element identified according to the method of claim 109.

131. An antisense molecule complementary to a genetic element identified according to the method of claim 109.

132. A method of inhibiting or activating RNAi in a cell comprising:

contacting the cell with a genetic element identified according to the method of claim 109.

133. A method for identifying cells containing a genetic element that inhibits or activates a miRNA translational repression pathway comprising steps of:

(a) providing a first population of mammalian cells members of which comprise (i) a nucleic acid that is integrated into the genome of the cell and provides a template for transcription of an mRNA transcript that encodes a detectable or selectable marker, wherein the mRNA transcript comprises one or more binding sites for an endogenous miRNA or an miRNA-like RNA; and

(ii) an endogenous miRNA or miRNA-like RNA that is expressed by the cell and represses translation of the mRNA that encodes a detectable or selectable marker;

(b) introducing a library into the population of cells, wherein the library comprises a plurality of genetic elements;

(c) identifying cells that display increased or decreased expression of the detectable or selectable marker relative to the starting population and do not display an alteration in mRNA transcript level sufficient to account for the increased or decreased expression of the marker, thereby identifying cells that contain a genetic element that inhibits or activates an miRNA translational repression pathway, respectively.

134. The method of claim 133, further comprising the step of identifying the genetic suppressor element.

135. A method for identifying a compound that inhibits or activates RNA interference comprising steps of:

(a) providing a population of mammalian cells members of which comprise a nucleic acid that encodes a detectable or selectable marker and express an RNAi-inducing agent that reduces expression of the detectable or selectable marker by RNA interference;

(b) contacting the cells with a compound; and

(c) identifying the compound as an inhibitor of RNAi if cells exhibit enhanced expression of the detectable or selectable marker after being contacted with the compound relative to cells not contacted with the compound or identifying the compound as an activator of RNAi if cells exhibit reduced expression of the detectable or selectable marker after being contacted with the compound relative to cells not contacted with the compound.

136. The method of claim 135, wherein the marker is a detectable marker.

137. The method of claim 135, wherein the marker is a selectable marker, and wherein enhanced expression of the selectable marker confers a growth advantage on cells expressing the marker under a selective condition, and wherein the step of identifying comprises exposing the contacted cells to the selective condition and isolating cells that survive or proliferate under the selective condition.

138. The method of claim 135, wherein the compound is a member of a compound library, and wherein the method comprises contacting a plurality of portions of the population with individual members of the compound libary and identifying one or more compounds as an inhibitor or activator of RNAi.

139. The method of claim 138, wherein the library is an annotated compound library.

140. The method of claim 138, wherein the library is a library of small molecules.

141. The method of claim 138, wherein the library is a natural product library.

142. The method of claim 138, wherein the library is a combinatorial library.

143. The method of claim 135, further comprising the step of:

performing a secondary screen or selection to retest the compound.

144. The method of claim 143, wherein the secondary screen or selection comprises:

(i) contacting a second population of mammalian cells members of which comprise a nucleic acid that encodes a second detectable or selectable marker and express an RNAi-inducing agent that reduces expression of the second detectable or selectable marker with the compound; and

(ii) assessing expression of the second detectable or selectable marker.

145. The method of claim 143, wherein the second marker is a detectable marker and the step of assessing expression of the marker comprises detecting the marker.

146. The method of claim 143, wherein the second marker is a selectable marker and the step of assessing expression of the marker comprises detecting cell growth under selective conditions.

147. The method of claim 135, further comprising the steps of:

(i) modifying the compound; and

(ii) testing the ability of the modified compound to activate or inhibit RNAi.

148. A compound identified according to the method of claim 135.

149. The compound of claim 148, further comprising an RNAi-inducing agent.

150. A method of inhibiting or activating RNAi in a cell comprising:

contacting the cell with the compound of claim 148.

152. The method of claim 152, wherein the compound is identified according to the method of claim 135.

151. A method of treating a subject comprising:

administering a therapeutic RNAi-inducing entity to the subject; and

administering a compound that activates an RNAi pathway to the subject.

153. A method for identifying a compound that inhibits or activates an miRNA translational repression pathway comprising steps of:

(a) providing a population of mammalian cells members of which comprise (i) a nucleic acid that is integrated into the genome of the cell and provides a template for transcription of an mRNA transcript that encodes a detectable or selectable marker, wherein the mRNA transcript comprises one or more binding sites for an endogenous miRNA or an miRNA-like RNA; and

(ii) an endogenous miRNA or miRNA-like RNA that is expressed by the cell and represses translation of the mRNA that encodes a detectable or selectable marker;

(b) contacting the cells with a compound; and

(c) identifying the compound as an inhibitor of a miRNA translational repression pathway if cells exhibit enhanced expression of the detectable or selectable marker after being contacted with the compound relative to cells not contacted with the compound and do not display enhanced mRNA transcript levels sufficient to account for the enhanced expression of the marker, or identifying the compound as an activator of an miRNA translational repression pathway if cells exhibit reduced expression of the detectable or selectable marker after being contacted with the compound relative to cells not contacted with the compound and do not display decreased mRNA transcript levels sufficient to account for the reduced expression of the marker.