mRNA maturation by two-step trans-splicing/polyadenylation processing in trypanosomes

Abstract

Trypanosomes are unique eukaryotic cells, in that they virtually lack mechanisms to control gene expression at the transcriptional level. These microorganisms mostly control protein synthesis by posttranscriptional regulation processes, like mRNA stabilization and degradation. Transcription in these cells is polycistronic. Tens to hundreds of protein-coding genes of unrelated function are arrayed in long clusters on the same DNA strand. Polycistrons are cotranscriptionally processed by trans-splicing at the 5' end and polyadenylation at the 3' end, generating monocistronic units ready for degradation or translation. In this work, we show that some trans-splicing/polyadenylation sites may be skipped during normal polycistronic processing. As a consequence, dicistronic units or monocistronic transcripts having long 3' UTRs are produced. Interestingly, these unspliced transcripts can be processed into mature mRNAs by the conventional trans-splicing/polyadenylation events leading to translation. To our knowledge, this is a previously undescribed mRNA maturation by trans-splicing uncoupled from transcription. We identified an RNA-recognition motif-type protein, homologous to the mammalian polypyrimidine tract-binding protein, interacting with one of the partially processed RNAs analyzed here that might be involved in exon skipping. We propose that splice-site skipping might be part of a posttranscriptional mechanism to regulate gene expression in trypanosomes, through the generation of premature nontranslatable RNA molecules.

Fig. 1.

Identification of a highly stable dicistronic pre-mRNA in the cytoplasm. (A) Northern blot of TcUBP RNA. Total RNA from the insect stage of the parasite was electrophoresed in an agarose gel and hybridized with the probes indicated below the images. (B) Schematic structure of the polycistronic gene cluster that contains TcUBP2 and TcUBP1 genes, the pre-mRNA unit, and the TcUBP1 and TcUBP2 monocistronic RNAs. (C) TcUBP RNA is localized in the cytoplasm. Total RNA (T), poly(A)+ RNA (pA+), and cytoplasmic (C) and nuclear (N) RNA fractions from insect-stage parasites were separated in an agarose gel and hybridized with the probes indicated below the images (schemes are not to scale). (D) TcUBP mRNA half-life was determined by ActD treatment. Insect-stage parasites were incubated with the drug, and total RNA was extracted at 0, 1, 2, 4, 6, and 8 h of treatment. Samples were separated on agarose gels and hybridized with the TcUBP probe. Northern blot signals were quantified by using Kodak (Rochester, NY) Image Software and plotted. The relationships between the percentage of mRNA remaining vs. time (hours in ActD) are shown. Molecular weight markers are shown on the right. The systematic gene name for TcUBP1 is Tc00.1047053507093.220 and for TcUBP2, Tc00.1047053507093.229.

Fig. 2.

TcUBP pre-mRNA is processed by trans-splicing. (A) Scheme of the pR-TcUBP DNA construct (dicistron with TcUBP1 CDS fused to GFP) made in pRIBOTEX vector. (B) Northern blot using RNA obtained from transfected parasites and hybridized with a GFP probe (Left); ∗ denotes the position of a band of ≈5 kb detectable when the film was overexposed. RT-PCR was performed by using total RNA extracted from pR-TcUBP transfected parasites. cDNA was synthesized by using a COOH-GFP primer, and the PCR was performed with TcSL and GFP as primers (see SI Table 3). The PCR products were separated in an agarose gel and visualized by ethidium bromide staining (Right). (C) Schematics of endogenous and recombinant TcUBP1 monocistronic mRNAs, showing that both share the same 5′ UTR. (D) Western blot was performed by using total protein extracts from transfected parasites (pR and pR-TcUBP) and probed with polyclonal rabbit anti-RNA-recognition motif sera (1/1,000 dilution). (E) Fluorescence microscopy of a T. cruzi insect-stage parasite transfected with the pR-TcUBP construct shown in A. 3′ SS, acceptor splice site; pPy, polypyrimidine tract; N, nuclear DNA; K, kinetoplast DNA. The molecular weight markers are shown on the right.

Fig. 3.

Trans-splicing/polyadenylation events in the 3′ UTR of TS RNAs. (A) Scheme showing the location of the probes used in these experiments. (B) Northern blots of poly(A)+ RNA from parasites treated with ActD for the indicated times. Hybridizations were performed with mTS-3′ UTR and SAPA probes. The asterisk indicates the detection of a new band after 1 h of ActD treatment. A β-tubulin probe was used for loading control. (C) Structure of mTS, the new polyadenylated mTS, and the SL-mTS-3′ RNAs generated by trans-splicing. Nucleotides where trans-splicing and polyadenylation events take place are indicated. 3′SS, 3′ trans-splicing sites; pPy, polypyrimidine tract. The drawings are not in scale. One representative sequence is shown. (D) Northern blots of total RNA from wild-type parasites (ctrol.) and parasites transfected with different pTEX constructs (D, TS with a 90-nt deletion including the pPy sequence and two 3′SS, R, TS in which the above-mentioned 90-nt fragment has been replaced by another sequence) without treatment (−) or incubated with ActD for 4 h (4). Hybridizations were performed with mTS-3′ UTR probe. The upper arrow indicates the SL-mTS-gapdh-3′ species. The lower arrow shows the endogenous SL-mTS-3′. (E) Structure of the recombinant mTS-gapdh mRNA and the SL-mTS-gapdh-3′ RNA generated by trans-splicing. 3′SS, acceptor splice site; pPy, polypyrimidine tract.

Fig. 4.

Stability and subcellular localization of mTS and iTS mRNAs. (A and D) Northern blots of total RNA from parasites treated with ActD for the indicated times. Hybridizations were performed with mTS-3′ UTR (A) and iTS-3′ UTR (D) probes. For the Northern blot made from mammalian-stage parasites, sequential hybridizations were performed by using the same blot after complete removal of the radioactive signal. Films were scanned, the signals quantified, and the ratio of TS RNAs to rRNA plotted (B and C for mTS and E for iTS). In the case of mTS, the larger RNA bands are indicated with open circles or closed triangles, and the smaller RNA bands are indicated with closed circles or open triangles (B and C). The SL-mTS-3′ RNA is hardly detectable in the mammalian stage, so we were able to quantify this RNA species only in the insect-vector parasite stage (B). (F) Subcellular localization of the different RNAs. Insect-stage parasites after 4 h of ActD treatment or without treatment were used to prepare nuclear (N) and cytoplasmic (C) fractions for RNA extraction. Hybridizations were performed with the indicated probes: 3′ UTR or SAPA probes for mTS and 3′ UTR probe for iTS. HSP70 and sn-250 mRNAs were used as controls for cytoplasmic and nuclear fractions, respectively. Arrows indicate small RNA products in the nuclear fraction.

Fig. 5.

Functional cis-acting elements in the noncoding regions of TcUBP dicistron interact in vivo with DRBD4 and HSP70. (A) Scheme of cis-acting elements predicted in the ICR and mTS-3′ UTR using bioinformatics tools. Polypyrimidine tracts, GA-rich elements, AU-rich elements, U-rich elements, and PTB sites are indicated. The numbers indicate putative PTB sites. The following numbers indicate the beginning and end positions of PTB sites within the ICR: UCUUC, 103–107, 106–110, 109–113, 817–821, 977–981, 1490–1494, 2356–2360, 3052–3056, 3055–3059, and 3169–3173; and CUCUCU, 4–9, 78–83, 80–85, 82–87, 795–800, and 797–802. Within the mTS-3′ UTR, the only putative PTB-binding site found was UCUUC, 288–292. (B) Agarose gel showing the RT-PCR products of TcUBP, mTS-3′ UTR, and Amastin from total RNA, coimmunoprecipitated in vivo with control rabbit serum (immunoprecipitation control, CTRL), anti-UBP1, anti-DRBD4, or -HSP70 antibodies. RT was performed with (+) or without (−) SuperScript II enzyme. For mTS-3′ UTR cDNA synthesis, we used oligod(T)₁₈. For TcUBP, the internal specific primer NH₂/AS-ubp1 (see SI Table 3) was used. Schemes of mRNAs with 3′ UTRs and position of primers used in PCRs are shown at the left. RT, reverse transcriptase enzyme.

Fig. 6.

Model of intermediate RNA maturation in T. cruzi. Two steps of trans-splicing and polyadenylation processing generate functional monocistronic mRNAs (see Discussion). In some cases, the second monocistron might not be translated into protein, as is the case for SL-mTS-3′ small RNA. ORF1, ORF2, ORF3, and ORF4, ORFs; pA, polyadenylation site; tsp, trans-splicing site.