tRNA gene sequences are required for transcriptional silencing in Entamoeba histolytica

Abstract

Transcriptional silencing by trans inactivation can contribute to the regulation of gene expression in eukaryotic cells. In the human intestinal protozoan parasite Entamoeba histolytica, trans inactivation of the amoebapore-A gene (AP-A) was recently achieved by episomal transfection of E. histolytica trophozoites with the plasmid psAP1. The mechanism of AP-A trans inactivation is largely unknown, though it was suggested that a partial short interspersed transposable element (SINE) is required. By systematic assessment of various E. histolytica isolates transfected with psAP1 derivates, trans inactivation of AP-A was restricted to the strain HM-1:IMSS (2411) but could not be achieved in other standard laboratory strains. Importantly, sequences of an E. histolytica tRNA array that were located on psAP1 in close proximity to the AP-A upstream region and comprising the glutamic acid (TTC) (E) and tyrosine (GTA) (Y) tRNA genes were indispensable for AP-A silencing. In contrast to the case described in previous reports, SINE was not required for AP-A trans inactivation. AP-A expression could be regained in silenced cells by episomal transfection under the control of a heterologous E. histolytica promoter, opening a way toward future silencing of individual genes of interest in E. histolytica. Our results indicate that tRNA gene-mediated silencing is not restricted to Saccharomyces cerevisiae.

Fig. 1.

trans inactivation of AP-A in the E. histolytica isolate HM-1:IMSS. The abundance of AP-A mRNA was analyzed in various E. histolytica isolates transfected with plasmid psAP1. Shown is an autoradiogram of a Northern blot from gel-separated total RNA isolated from nontransfected (wild-type) or psAP1-transfected amoebae of HM-1:IMSS (2411) or the ATCC HM-1:IMSS strain. The blot was sequentially hybridized with radiolabeled AP-A and E. histolytica ACTIN gene probes. Note that trans inactivation was achieved in HM-1:IMSS (2411) but not in other E. histolytica isolates.

Fig. 2.

Reconstitution of AP-A expression in AP-A trans inactivated cells by episomal transfection with plasmid pNinAP-A. (A) Schematic depiction of the expression vector comprising the neomycin resistance gene (NEO) under the control of E. histolytica actin (ACT) gene 5′ and 3′ sequences and the AP-A open reading frame (AP-A) under the control of E. histolytica lectin gene 5′ (LEC 5′) and E. histolytica actin gene 3′ (ACT 3′) sequences. The lectin gene promoter and the AP-A open reading frame are separated by an intron from the gene encoding E. histolytica ribosomal protein L27a. (B) Immunoblot analysis of E. histolytica extracts from various HM-1:IMSS (2411) transfectants tested with antisera against E. histolytica AP-A or AP-B. As a control, a parallel blot was developed with antiserum against E. histolytica superoxide dismutase (SOD). Extracts were obtained from the following transfectants: wild-type cells transfected with pN (neocassette vector) and selected with G418; wild-type cells transfected with psAP1; AP-A-silenced cells grown for several months without G418 and subsequently transfected with pNinAP-A and selected again with G418 (pNinAP-A+G418) or without G418 selection (pNinAP-A-G418); and wild-type cells transfected with psAP1Δ2600. Note that reconstitution of expression was achieved when selective pressure was maintained after transfection with pNinAP-A.

Fig. 3.

An E. histolytica tRNA array is required to mediate trans inactivation of AP-A. (A) Northern blot analysis of gel-separated total RNA isolated from various amoeba transfectants as indicated. The blot was hybridized with radiolabeled ACTIN and AP-A gene probes. (B and C) Schematic depiction of sequence elements present on psAP1 and on various deletion constructs used for transfection. Compared to psAP1, plasmids pNAP-A and psAP1Δ2600 bear deletions of 3.2 and 2.6 kb, respectively, as indicated by the dotted lines. Transfection plasmids pNAP-AEY1000 and psAP1Δ2600EY1000 represent pNAP-A and psAP1Δ2600, respectively, in which the 1.0-kb EY tRNA array has been introduced. AP-A mRNA abundance was measured by qRT-PCR with primers specific for the E. histolytica AP-A and actin (ACT) genes, and the ΔΔCT method was used for quantification. The mRNA abundance of wild-type HM-1:IMSS (2411) amoebae was set at 1. A value higher than 1 indicates overexpression, while a value of 0 indicates that AP-A was trans inactivated. Note that only those transfection plasmids containing the EY tRNA array have AP-A-transinactivating capacity. NEO, neomycin resistance gene. hypoth., hypothetical protein.

Fig. 4.

Sequence motifs within the EY tRNA array are required for AP-A trans inactivation. (A) Transfection plasmid psAP1Δ2600, incapable of mediating trans inactivation of AP-A, was used as a backbone to introduce various modifications of the 1.0-kb EY tRNA array as indicated. Glutamic acid (E) and tyrosine (Y) tRNAs are represented by arrows, and AT-rich repeat elements are shown as dashes. NEO, neomycin resistance gene. (B) AP-A mRNA abundance was measured by qRT-PCR with primers specific for AP-A or actin and the ΔΔCT method for quantification. To the right, AP-A mRNA abundances relative to that of wild-type cells (set at 1) are given. The relevance of polythymidine stretches (TTTTT) adjacent to the tRNA sequences was investigated. Note that trans inactivation was observed only with constructs containing both tRNA sequences but was independent of the presence of poly(T) stretches or repeat elements.

Fig. 5.

The presence but not transcription of the tRNA on psAP1 is required for AP-A trans inactivation. (A) Schematic depiction of the relevant regions of psAP1 and location of various mutations introduced into the tyrosine (Y) tRNA gene to test the importance of the respective residues for the trans inactivation capacity of psAP1. Residues deviating from the wild-type sequence (Y wt) are underlined (Y mut). (B) Presence or absence of AP-A mRNA and tRNA transcripts after transfection with psAP1 containing mutations within the tyrosine tRNA gene. The abundances of AP-A mRNA and transcribed mutated tRNA relative to that of the wild type (set to 1), as determined by qRT-PCR, are given. Note that mutated tRNA was detectable only when mutations indicated by asterisks in panel A were introduced (Y tag1) but not when bearing mutations marked by arrows in that panel. The latter primarily comprised mutations located within the control region (A-box and B-box), which are likely to affect binding capacity of the polymerase III transcriptional machinery. However, none of the mutations affected AP-A trans inactivation capacity, indicating that the presence of the tRNA gene, but not its transcription or binding of the transcriptional machinery, is required for trans inactivation. NA, not applicable.

Fig. 6.

SINE and a putative PD1 element are dispensable for AP-A trans inactivation. The silencing plasmid psAP2 containing largely the same sequence elements as psAP1 but lacking the AP-A coding and 3′ flanking sequences was used to analyze the requirement for SINE and the putative PD1 element for AP-A trans inactivation. Various 5′ deletion mutant forms of the AP-A upstream region were generated and substituted for the AP-A 5′ region in psAP2 as indicated. Even the minimal fragment tested (psAP2Δ260nt) comprising 213 bp of the AP-A upstream region but lacking SINE as well as PD1 mediated trans inactivation. Note that in contrast to the longer fragments, which silenced AP-A in 100% of transfected amoebae, deletion plasmids psAP2Δ240nt and psAP2Δ260nt induced trans inactivation efficiency in only 50% and 20% of transfected cells, respectively. NEO, neomycin resistance gene.