TblncRNA-23, a long non-coding RNA transcribed by RNA polymerase I, regulates developmental changes in Trypanosoma brucei

Galili-Kostin, Beathrice; Rajan, K. Shanmugha; Ida Ashkenazi, Yuval; Freedman, Almog; Doniger, Tirza; Cohen-Chalamish, Smadar; Waldman Ben-Asher, Hiba; Unger, Ron; Roditi, Isabel; Tschudi, Christian; Michaeli, Shulamit

doi:10.1038/s41467-025-58979-w

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Published: 18 April 2025

TblncRNA-23, a long non-coding RNA transcribed by RNA polymerase I, regulates developmental changes in Trypanosoma brucei

Nature Communications volume 16, Article number: 3697 (2025) Cite this article

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Abstract

The protozoan parasite Trypanosoma brucei undergoes a complex life cycle, moving between its mammalian host and the blood-feeding tsetse fly vector. The two major surface proteins expressed by procyclic forms in the insect midgut, EP and GPEET procyclin, are transcribed from a polycistronic transcription unit by RNA polymerase I. Here we identify a long non-coding RNA, TblncRNA-23, that is encoded between the two procyclin genes. TblncRNA-23 localizes to the nucleolus and also associates with polysomes. Overexpression of TblncRNA-23 and its down regulation by RNAi or knockout (KO) identify EP and GPEET mRNAs as targets, among other mRNAs that changed abundance in the transition from early to late procyclic forms and from procylic to the metacylic forms, suggesting its role in regulating gene expression which accomapines or dictates of the parasite transitions within in its insect host. TblncRNA-23 interacts with its substrates via base-pairing using different domains. Purification of TblncRNA-23-associated proteins by RaPID identifies hundreds of proteins, including proteins translated from its target mRNAs, suggesting its association with translating ribosomes. Early and late procyclic forms differ in their social motility (SoMo) capabilities, which is essential for migration away from the insect midgut to enable parasite transmission. Overexpression of TblncRNA-23 results in hypermotility, whereas KO compromises this capacity, suggesting a regulatory role in SoMo. Moreover, silencing of the RNA abrogates the ability of the parasite to transform from procylic to the metacyclic forms affecting the parasite’s potential to cycle between its hosts.

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Introduction

The parasite Trypanosoma brucei causes sleeping sickness in humans and Nagana in domestic animals. The parasite cycles between a mammalian host and the tsetse fly. In the mammalian host, parasites propagate as slender bloodstream forms (BSF). Upon ingestion by the fly, the parasite differentiate into early procyclic forms (PCF) in the lumen of the insect midgut¹. In addition to major changes in morphology and metabolism, parasites replace the variant surface glycoprotein (VSG) with two insect-specific coat proteins known as GPEET and EP procyclins². At this point in the infection cycle, there are two possible outcomes. Either the early PCF is eliminated by the fly, or the parasites reach the other side of the peritrophic matrix and colonize the ectoperitrophic space. Once the infection is established, parasites in the midgut are considered to be late PCF², which express EP, but not GPEET². Transcriptomic analysis of early and late PCF in culture identified 73 transcripts that are differentially regulated³ between the early and late stages. In addition, a phenotypic difference between early and late PCF is their ability to engage in social motility (SoMo) when the parasites are plated on semi-solid plates⁴. This motility was recently shown to comprise a chemotactic response to self-generated pH gradients⁵. To complete the life cycle in the fly, parasites undergo further rounds of differentiation and move to the proventriculus, where they transform into epimastigotes and then colonize the salivary glands, culminating in the production of metacyclic forms, which can be transmitted to a new mammalian host¹. Overexpression of the RNA binding protein RBP6 in PCF induces their transformation to metacyclic forms⁶ and this transition involves changes in the expression of hundreds of transcripts^7,8. An RNAi screen identified factors that are essential for the transition to either epimastigotes or to metacyclic forms⁹.

GPEET and two isoforms of EP (EP1, EP3) are marker proteins for the transition from BSF to early and late PCF. The 3’ UTR of both mRNAs has a conserved structure that is composed of three stem-loops (LI-III) that play a role in stage-specific expression^10,11,12,13. Two domains, a 26-mer present in LII and a 16-mer present in LIII, are conserved in all EP and GPEET 3’ UTRs^10,13. The 26-mer is a destabilizing element in all life-cycle stages. The 16-mer is implicated in RNA stability and translational control¹⁰. TbZFP3 is the only RNA binding protein that was shown to date to bind LII in EP1, and regulates translation by competing out one or more unidentified repressor proteins¹⁴.

EP and GPEET mRNAs are derived from a polycistronic RNA precursor transcribed by RNA polymerase I (pol I)^15,16. The level of these transcripts is reduced to almost undetectable levels in the BSF¹⁰. This downregulation is controlled by three mechanisms: transcriptional down-regulation, rapid degradation of the mRNA, and translational regulation¹⁰.

Apart from EP and GPEET, which are transcribed by pol I¹⁷, all other mRNA coding genes are constitutively transcribed by pol II forming polycistronic units which are processed by the concerted action of trans-splicing and polyadenylation¹⁸. The differential gene expression of transcripts from the same polycistronic transcription unit, as well as stage-specific gene expression, is mediated by hundreds of RNA-binding proteins¹⁹.

Little is known about non-coding RNAs that regulate gene expression in trypanosomes²⁰. These parasites appear to lack microRNAs²¹. However, our group recently identified abundant ncRNAs that are developmentally regulated and/or respond to stress²². RNA sequencing led to the identification of 1428 previously uncharacterized lncRNA transcripts. One of these is the grumpy lncRNA²³. A small nucleolar RNA is processed from this lncRNA to form the snoGRUMPY, which binds to mRNAs and regulates their expression. Grumpy overexpression reduces parasitemia in infected mice²³.

In this study, we reveal the function of a pol I-transcribed lncRNA that regulates the expression of genes associated with the transition from early to late procyclic forms and from procyclic to metacyclic forms, identify its targets, and demonstrate how it controls social motility of these parasites.

Results

TblncRNA-23 is a long-noncoding RNA transcribed by RNA pol I

A screen conducted to identify novel ncRNAs in T. brucei identified TbsRNA-23, which is preferentially expressed in PCF²². Analysis of RNA-seq data of polysomal (translating) and sub-polysomal (non-translating) mRNAs²⁴ revealed that TbsRNA-23 was encoded within the procyclin loci located on chromosomes 6 and 10 (Fig. 1A) and that it is a long non-coding RNA. We therefore changed its name to TblncRNA-23. The procyclin loci are transcribed by RNA pol I^16,25 into a polycistronic RNA precursor, giving rise to EP and GPEET mRNAs, and TblncRNA-23 which is trans-spliced and polyadenylated²⁴. It is currently unknown if pol II is also involved in transcription within this locus. Inspection of its expression in transcriptomes obtained from parasites during infection in the fly, and in culture-derived slender and stumpy BSF showed that TblncRNA-23 expression is highest during midgut infection (days 7-28), but it is not detectable in BSF (Fig. 1B)^3,26. Its expression was verified by Northern blot analysis (Fig. 1C). The exact size of 404 bases was determined from its sequence in polyA primed libraries²⁷ (Supplementary Fig. 1). The localization of TblncRNA-23 was determined by in situ hybridization combined with immunofluorescence using antibodies against the nucleolar protein, NHP2 (Fig. 1D)²⁸, and suggested that TblncRNA-23 RNA is localized in the nucleolus, where EP and GPEET were shown to be transcribed²⁹. TblncRNA-23 distribution was examined on RNPs separated by sucrose-gradient centrifugation that revealed one major peak that is larger in size than snoRNPs ( ~10S), as well as two less abundant peaks. Based on the fractionation of TB11Cs2C2, a snoRNA involved in rRNA processing³⁰, a minor fraction of TblncRNA-23 appeared to co-migrate with rRNA-processing complexes, as well with polysomal fractions where translating GPEET mRNA fractionated (Fig. 1E). Despite the localization of the TblncRNA-23 in the cytoplasm on translating ribosomes, a signal was not observed in the cytoplasm by in situ hybridization, most likely because it is not as concentrated as in the nucleolus.

Fig. 1: Genomic, transcriptomics, and cellular localization of TblncRNA-23. — Fig. 1: Genomic, transcriptomics, and cellular localization of *TblncRNA-23.*

Interactions of TblncRNA-23 with GPEET and other mRNAs

We next determined whether TblncRNA-23 interacts with mRNAs. To this end, we used interactomes that were prepared from cells exposed to psoralen (AMT) and subjected to UV irradiation to crosslink interacting RNA molecules²². The chimeric molecules containing TblncRNA-23 are presented in Fig. 2A. We observed cross-linking between TblncRNA-23 and the EP-coding region, and with the GPEET 3’ UTR. Additional mRNAs were identified as potential substrates (Fig. 2A). The levels of these mRNAs changed upon overexpression of TblncRNA-23 or its silencing, suggesting that they are regulated by TblncRNA-23 (see below). To examine if EP and GPEET are regulated by TblncRNA-23, the lncRNA was targeted by dsRNA, which resulted in ~80% reduction in the level of the RNA (Fig. 2B i). To achieve a better depletion of the RNA, the gene was knocked out using CRISPR-Cas9. Knockout (KO) resulted in the complete elimination of cellular TblncRNA-23, suggesting that the gene was removed from both copies of chromosome 6 and chromosome 10 (Fig. 2B ii). TblncRNA-23 silencing led to a concomitant reduction in the level of GPEET protein (Fig. 2C i) and an increase in the expression of EP protein (Fig. 2C ii). Likewise, KO also led to a reduction in the level of GPEET (Fig. 2E i). In contrast, the EP protein level was not increased as expected, but was reduced to 40% of its level in the control and its migration was retarded compared to the parental strain (PS) (Fig. 2F i). However, the level of EP mRNA was increased as expected (Fig. 2f ii). The complete elimination of TblncRNA-23 in the KO may have affected the protein’s post-translational modification, altering its stability.

Fig. 2: EP and GPEET levels are regulated by TblncRNA-23. — Fig. 2: EP and GPEET levels are regulated by *TblncRNA-23.*

To further examine if the changes in expression were due to changes in the levels of mRNA and/or regulation of translation, the levels of GPEET and EP mRNA were determined by Northern analyses (Fig. 2D i and ii). While the reduction in GPEET protein could be explained almost entirely by the change in mRNA level, the EP protein level was increased by 6-fold and that of mRNA by 4-fold, suggesting that enhanced translation may also contribute to its increase. The changes in GPEET and EP levels, which are inversely correlated, resemble the changes that take place during the transition from early to late PCF, when GPEET mRNA and proteins decrease, and EP mRNA and proteins increase^2,31.

TblncRNA-23 interacts with GPEET mRNA by base-pairing with its 3’ UTR, and the interaction domain is essential for its regulation

Bioinformatic analysis suggested a potential for base pairing between TblncRNA-23 and L II of the GPEET 3’ UTR (Fig. 3A i). To further study the validity of the interaction domain, TblncRNA-23 was tagged to monitor its expression by inserting a boxB (15 nt) at position 133 (Fig. 3A ii). Three types of mutations were introduced into the tagged TblncRNA-23 gene (Fig. 3A ii): a mutation removing the entire interaction domain, and two substitution mutations. Cells overexpressing wild-type and mutated TblncRNA-23 were generated, and a ~ 3-fold and 2.5-fold increase in the level of wild-type and mutant transcripts, respectively, were observed (Fig. 3B). Overexpression (OE) of the wild-type TblncRNA-23 induced a > 10-fold increase in the level of GPEET protein, and the EP protein was reduced to undetectable levels (Fig. 3C). Deletion of the interaction domain in TblncRNA-23 abolished the ability of the lncRNA to increase GPEET or reduce EP proteins. The two substitution mutations increased the level of GPEET 7-fold, with the EP protein at undetectable levels (Fig. 3C). These data suggested that the interaction domain identified by bioinformatics is valid and sufficient to control GPEET expression. Note that deleting the interaction domain of TblncRNA-23 with the GPEET mRNA drastically increased the accumulation of EP protein, suggesting that this domain might also be important for regulating EP by an as yet unknown mechanism.

Fig. 3: TblncRNA-23 regulates the level of GPEET and EP by interacting with their 3’ UTR. — **Fig. 3: *TblncRNA-23* regulates the level of GPEET and EP by interacting with their 3’ UTR.**

To examine if the regulation by TblncRNA-23 is mediated by the 3’ UTR of both interacting mRNAs, we used a reporter construct in which the coding sequence of glutamic acid alanine rich protein (GARP) was ligated to different versions of the EP or GPEET 3’ UTR^2,10. Expressing the entire GPEET 3’ UTR led to a 2-fold increase in the level of GARP when TblncRNA-23 was overexpressed (Fig. 3D), like the effect on the GPEET gene (Fig. 2). However, the LII domain alone was not sufficient to increase the expression of GARP, suggesting that additional 3’ UTR sequences are necessary. Expressing GARP linked to the 3’ UTR of EP led to a marked decrease in GARP when TblncRNA-23 was overexpressed (Fig. 3D), similar to the regulation pattern of the native EP (Fig. 2). This phenotype could not be achieved when LII was absent from the 3’ UTR. In fact, the level of GARP increased slightly, suggesting that, as in the case of GPEET, LII is important for its regulation by TblncRNA-23 (Fig. 3D). However, it is not sufficient to repress the expression of GARP, suggesting that the regulation involves additional domains of the 3’ UTR.

TblncRNA-23 overexpression and downregulation regulate additional mRNAs that are differentially expressed in early and late PCF

The regulation of EP and GPEET observed upon overexpression of TblncRNA-23 resembles the expression of these proteins in early and late PCF^3,26. To examine if TblncRNA-23 regulates additional genes involved in this transition, we determined the transcriptome of cells overexpressing TblncRNA-23. We identified 830 transcripts (out of 10,586) whose levels were significantly changed (p value ranging from 10^-160-10^-2) by 1.5- to 12- fold between parental strain and overexpressing cells (Supplementary Data 1). Among the dysregulated transcripts are 74 that are differentially expressed in early and late PCF²⁶ (Supplementary Data 1), and 283 are transcripts whose levels changed during the RBP6-induced transition from PCF to metacyclic forms (marked in yellow)^32,33. A volcano plot illustrates the changes between PS and the cells OE TblncRNA-23, highlighting transcripts that were dramatically increased (Fig. 4A). Gene ontology (GO) of these transcripts showed a connection to the process of social motility (SoMo), such as cAMP signaling^34,35,36 (Supplementary Fig. 5). Among the 74 transcripts that were previously shown to change from early to late PCF^3,26, 46 transcripts that were increased in early PCF were elevated in the TblncRNA-23 OE cell line and among 26 transcripts that were shown to be up-regulated in late PCF^3,26 17 were downregulated in TblncRNA-23- OE cells (Fig. 4B). We examined the significance of these changes using Chi-squared test and the significant values of 490 (p value = 9.4 e-109) was obtained for genes elevated in early PCF and 114 (p value = 1.04e-26) for genes that were elevated in late PCF. To further validate these findings, we examined the expression of thymidine-7-hydroxylase (TLP7) and found that TblncRNA-23 has the potential to interact with its 5’ UTR (Fig. 4C i). This interaction is supported by the presence of a chimeric RNA species identified in the interactome (Fig. 4C i). Indeed, the level of TLP7 mRNA was increased ~three-fold upon TblncRNA-23 overexpression (Fig. 4C ii). Another example of TblncRNA-23 interaction with 3’ UTRs are with putative pteridine transporter genes encoded on chromosome 1 (exemplified by Tb927.1.2880). The interaction between TblncRNA-23 and the pteridine transporter is also supported by a chimeric molecule between these RNA molecules (Fig. 4D i). Overexpression of TblncRNA-23 reduced the mRNA level, while a mutation introduced into the interaction domain abolished this response (Fig. 4D ii). Taken together, these data suggested that TblncRNA-23 is likely to regulate gene expression associated with the transition from early to late PCF (see below).

Fig. 4: TblncRNA-23 regulates gene expression involved in early to late PCF transition and interacts with its substrates via different domains. — **Fig. 4: *TblncRNA-23* regulates gene expression involved in early to late PCF transition and interacts with its substrates via different domains.**

Since lncRNA are known to regulate expression at the level of both mRNA stability and translation, we examined the proteome of cells expressing TblncRNA-23 by isotope dimethyl labeling. This method is quantitative, does not require the in vivo incorporation of the isotope, and is fast and efficient³⁷. Analysis of the proteome shows that the levels of 36 proteins were significantly changed (Supplementary Data 2). For 17 proteins, the change in the protein level was consistent with a change in the mRNA level. Among these, 9 are developmentally regulated in early or late PCF (Fig. 5A). Nineteen proteins were affected only at the protein level, and these may be encoded by mRNAs whose translation is regulated by TblncRNA-23. To gain additional support for the effect of TblncRNA-23 on gene expression, the transcriptome of TblncRNA-23-silenced cells were determined after 2.5 days of silencing by RNA-seq of polyA RNA. We identified 675 and 698 transcripts that were up-regulated or down-regulated, respectively, by at least 1.5-fold (p value ≤ 10^-2). The transcripts that were shown to be inversely regulated when comparing OE to silenced cells are presented in a heat map (Fig. 5B, Supplementary Data 3). This demonstrates that among 248 transcripts detected in both transcriptomes, 185 transcripts were inversely affected as expected, whereas only 65 transcripts were affected in the same way. In summary, at least ~200 transcripts are directly affected by the level of TblncRNA-23.

Fig. 5: TblncRNA-23 regulates its substrates at the level of mRNA stability and translation. — **Fig. 5: *TblncRNA-23* regulates its substrates at the level of mRNA stability and translation.**

Identification of proteins associated with TblncRNA-23 using RNA-protein interaction (RaPID) assays

To identify protein factors that bind to TblncRNA-23, we used the RaPID methodology. The boxB stem-loop was placed at position 133 of TblncRNA-23, and this addition did not interfere with the ability to overexpress the RNA (Fig. 3B). Proteins that associated with the tagged TblncRNA-23 become biotinylated and the purified proteins were analyzed by LC-MS (Supplementary Data 4). To identify proteins that are specifically associated with the RNA, the enrichment of each protein was calculated³⁸; 392 proteins were significantly enriched (Supplementary Data 4). The variability among experiments reflects variation in the efficiency of purification and/or MS analysis. We performed the purification seven times using whole cell extracts and selected to present four of the purifications that enriched the same proteins at levels of ten- to hundred-fold in at least three out of four experiments. Among these 51 proteins were selected in four purifications. The enriched proteins either directly bind to TblncRNA-23 or are present in complexes bound by it. (Supplementary Data 4). The selected proteins were enriched for ribosomal proteins and translation factors as well as with pre-mRNA processing proteins and rRNA processing factors. Many metabolic enzymes were also identified. Such enzymes were shown previously to associate with active ribosomes in mammals³⁹ as well as in trypanosomes⁴⁰. We identified 38 proteins that are encoded by mRNAs regulated by TblncRNA-23 (highlighted in Supplementary Data 4). Thus, RaPID appears to identify ribosomes that translate TblncRNA-23 substrates. The TblncRNA-23 purification also identified well-defined RNA-binding proteins, known to regulate mRNA expression⁴¹ that either bind directly to TblncRNA-23 or to its mRNA substrates (Fig. 6A). Since TblncRNA-23 was localized to the nucleolus, high salt nuclear extracts were prepared and used for the purification. Such purifications should exclusively identify the TblncRNA-23 nuclear complexes. Among the proteins identified by LC-MS in the two biological replicates (r = 0.75) 150 proteins were nuclear (Supplementary Data 5) and ~100 were specific only to the nuclear extract, most likely because of the high salt extraction. These include proteins involved in rRNA processing, chromatin remodeling and nucleopore factors (Fig. 6B, Supplementary Data 5).

Fig. 6: RapID of tagged TblncRNA-23 identified its binding proteins and associated complexes. — **Fig. 6: RapID of tagged *TblncRNA-23* identified its binding proteins and associated complexes.**

PUF7 binds to TblncRNA-23 and regulates many of its mRNA substrates

One of the proteins that was enriched in the RaPID purification was PUF7, which was shown to form complexes with NRG1 (nucleolar regulator of GPEET), PUF10 and BOP1⁴². The silencing of these transcripts prolonged or increased GPEET expression⁴². Using sucrose gradient fractionation, we detected tagged PUF7, but not NRG1 protein in fractions containing TblncRNA-23 (see Figs. 1E and 6C), suggesting that NRG1 is part of a complex that binds to TblncRNA-23 only in the nucleolus, as opposed to PUF7, which is present in both complexes (Fig. 6C). Direct binding of PUF7 was examined using PTP-tagged protein. The RNA from each sample (total, IgG-bound and supernatant) was examined by real-time PCR using TblncRNA-23 primers. The ratio presented reflects the normalized level of TblncRNA-23 with respect to tubulin. The results showed a 15-fold enrichment of TblncRNA-23 on the beads (Fig. 6D). TblncRNA-23 binds PUF7 and is likely to donate or remove it from its mRNA substrates. Such substrates should be co-regulated by both PUF7 and TblncRNA-23. To identify these substrates the transcriptome of the PUF7-silenced cells was determined. We detected 9,673 transcripts (R = 0.9), of which about 300 transcripts were dysregulated (p < 0.01), with 80 affected both by silencing of PUF7 or by overexpressing TblncRNA-23 (Supplementary Data 6). In many cases, overexpression of TblncRNA-23 increased the levels of these mRNAs, and the same mRNAs were also increased by silencing of PUF7, suggesting TblncRNA-23 removes PUF7 from its mRNA target leading to their stabilization like under PUF7 silencing. In addition, 28 transcripts were inversely affected by TblncRNA-23 silencing i.e. reductions in their levels, suggesting that in the absence of TblncRNA-23 PUF7 is not removed from the mRNA targets and these are therefore destabilized. In other cases, the opposite effect was found, i.e. silencing of PUF7 reduced the mRNA level, suggesting that in these cases PUF7 may stabilize these mRNAs. Thus, TblncRNA-23 functions in modulating the binding of PUF7 to many of its targets and by that controls the fate of these mRNAs.

Overexpression or knockout of TblncRNA-23 affects social motility in early PCF parasites

It was previously shown that early PCF exhibit social motility when plated on a semi-solid surface⁴³. To ensure that the overexpression of GPEET and reduction in EP is shared by individual cells in the community, flow cytometry was performed using EP and GPEET antibodies. The results showed that many of cells overexpressing TblncRNA-23 highly expressed GPEET, but showed a reduction in EP (Fig. 7A). The PS and an isogenic strain OE TblncRNA-23 were spotted (2 x 10⁶ cells) on agarose plates. Both cell populations migrated and formed projections (Fig. 7B, Supplementary Fig. 7 i). Cells OE TblncRNA-23 exhibited hyperSoMo (Fig. 7B, Supplementary Fig. 7 i). The communities on the plates were then transferred to nitrocellulose and reacted with antibodies to GPEET and EP. In PS, the late PCF were located in the center of the colony, and cells expressing GPEET were detected at the edges of the projections. In contrast, many more cells in the TblncRNA-23 OE population expressed GPEET, with the exception of a small population in the center that expressed mainly EP (Fig. 7B, Supplementary Fig. 7 i). Moreover, the projections at the edges of colonies were bifurcated. These results suggested that OE of TblncRNA-23 induced the cells to remain at the early PCF stage with active SoMo. The effect on SoMo was examined in the KO strain. The results (Fig. 7C, Supplementary Fig. 7 ii) demonstrated that the KO cells are severely compromised in performing SoMo, compared to PS, but not completely negative. The KO population expressed less GPEET as demonstrated in (Fig. 2E), suggesting that TblncRNA-23 regulates gene expression, which is essential for the early to late PCF transition.

Fig. 7: TblncRNA-23 regulates social motility. — **Fig. 7: *TblncRNA-23* regulates social motility.**

TblncRNA-23 is required for differentiation to metacyclic forms

The OE and silencing (Supplementary Data 1, 3) showed effects on mRNAs involved in the transition to the metacyclic form, based on the transcriptome of cells OE RBP6^6,7. Indeed, significant enrichment using the Chi-squared test (356 p value 7.910591e-81) of transcripts involved in metacyclic transformation was found. To examine if TblncRNA-23 also participates in controlling this transition, we OE RBP6 in cells silenced for TblncRNA-23 and examined if silencing affected the ability of the parasites to transform to epimastigote and metacyclic forms. Epimastigotes reposition the kinetoplast anterior to the nucleus and express brucei alanine-rich protein (BARP)⁴⁴. Upon their transformation to metacyclic forms, the kinetoplast moves to the posterior end of the cell⁶. Microscopic examination of the kinetoplast position in cells either OE RBP6 or OE RBP6 and silenced for TblncRNA-23 for 5 days is presented in (Fig. 8A) and illustrates the difference in localization of the kinetoplast upon induction. Epimastigotes were found upon RBP6 OE and also in cells OE RBP6 that were silenced for TblncRNA-23. To confirm that the cells transformed to epimastigotes, we examined the expression of BARP⁴⁴. The results (Fig. 8B) confirmed BARP induction in cells OE RBP6 and silenced for TblncRNA-23. Quantification of microscopic experiments recorded after 4 days of induction indicated that 14% of cells transformed to epimastigotes upon RBP6 OE, but more than 80% were transformed to this stage in OE RBP6 cells and silenced for TblncRNA-23. Eight days after induction ~30% of the OE RBP6 cells were metacyclics, whereas in cells that were also silenced for TblncRNA-23 the transition to the metacyclic form was halted (Fig. 8C). Comparing the growth of cultures (Fig. 8D) demonstrated that the OE RBP6 cells grew almost normally but OE RBP6 cells silenced for TblncRNA-23 stopped growing. Next, we determined the transcriptome of the OE RBP6 cells silenced for TblncRNA-23 for 5 days and compared it to the transcriptome of OE RBP6 cells^7,8. The transcriptome of RBP6 (OE) revealed changes in 434 transcripts compared to uninduced cells^6,7. The transcriptome analysis of cells OE RBP6 and silencing for TblncRNA-23 showed effect on 62 transcripts that their level was inversely expressed when comparing their level in RBP6 OE cells (Supplementary Data 7). Note that it was sufficient to alter the expression of a single mRNA to stop developmental progression⁹. Thus, TblncRNA-23 also controls the expression of genes that are associated with and/or necessary for the transition from epimastigote to the metacyclic form.

Fig. 8: TblncRNA-23 regulates the transformation into the metacyclic form. — **Fig. 8: *TblncRNA-23* regulates the transformation into the metacyclic form.**

Discussion

Most lncRNA species described in eukaryotes to date are transcribed by pol II. This study identified a lncRNA, TblncRNA-23, that is derived from a polycistronic pol I transcript. TblncRNA-23 is produced in the nucleolus and migrates to the cytoplasm where it associates with polyribosomes, translating its targets, affecting their mRNA stability and translation. RaPID of TblncRNA-23 identified hundreds of proteins, including pre-rRNA processing factors, ribosome-associated proteins, and dozens of RNA-binding proteins that likely regulate TblncRNA-23 substrates. The levels of hundreds of mRNAs were affected upon TblncRNA-23 silencing or overexpression, and peptides encoded by tens of these mRNAs were detected, suggesting that TblncRNA-23 regulates these mRNAs during their translation. PCF cells OE or silenced for TblncRNA-23 differentially express mRNAs that are changed during the transition from early to late PCF and upon development to the metacyclic form, and thus, TblncRNA-23 regulates gene expression associated with transition from one life-cycle stage to the next.

Previous studies showed that the clusters of genes containing GPEET and EP, are transcribed by polymerase I⁴⁵, but the lncRNA regulating their expression had not been reported to date. The transcription of EP was also shown to take place in the nucleolus^29,45, supporting the localization we observed for TblncRNA-23 (Fig. 1D). GPEET and EP were shown to be regulated at the mRNA level mostly by the LII domain located in the 3’ UTR of both mRNAs^10,11,12,13. The interaction of TblncRNA-23 and GPEET was mapped to the region partially overlapping with LII in the 26-mer domain (Fig. 3a i). In contrast to the GPEET-TblncRNA-23 interaction, we could not identify by bioinformatics any potential interaction with EP mRNA, which might be discontinuous.

In this study, we show the involvement of PUF7 in regulating mRNA levels. Notably, PUF7 was shown to bind TblncRNA-23 (Fig. 6) and to regulate GPEET, and may compete with TblncRNA-23 for GPEET binding. PUF7 binding to GPEET results in its destabilization⁴². Overexpression of TblncRNA-23 may sequester PUF7, thereby preventing its binding to GPEET mRNA. Indeed, overexpression of TblncRNA-23 enhanced the expression of GPEET, similar to the effect on GPEET upon PUF7 silencing (Fig. 6). PUF7 is likely not the only protein that directly binds to TblncRNA-23. The other mRNA-binding proteins identified might be enriched due to their binding to mRNA targets of TblncRNA-23.

TblncRNA-23 may function at two regulatory levels, mRNA stability discussed above, and translation. Several translation factors were detected in the RaPID purification. Based on proteome analysis, 19 mRNAs were shown to be regulated solely at the level of translation (Supplementary Data 2), suggesting that TblncRNA-23 regulates translation, as well.

TblncRNA-23 was shown to interact with the 5’ and 3’ UTRs of mRNAs but via different domains (Fig. 3C and Fig. 4D). The interaction with the different substrates may deliver RNA binding protein(s) to these mRNA substrates. Alternatively, the binding of TblncRNA-23 to its substrate may create landing pads for RNA binding proteins that do not recognize either RNA individually. Studies from mammalian cells revealed a similar phenomenon. A lncRNA abundant in human skeletal myoblasts, associates via base-pairing with MEF2C mRNA, and stabilizes it by recruiting HuR to the MEF2C 3’ UTR, increasing MEF2C production and affecting myogenesis⁴⁶. An example that resembles the regulation of GPEET by PUF7 in sequestering decay-promoting RBP is the abundant lncRNA, NORAD. This lncRNA contains many binding sites for Pumilio (PUM1/2), an RNA binding protein, like PUF7 that typically reduces the stability and or translation of its target mRNAs⁴⁷. The sequestration of Pumilio by NORAD enables the production of several proteins involved in genomic stability⁴⁷. Specific effects on translation, similar to those observed for TblncRNA-23 were reported with LincRNAp21, which was shown to interact via base-pairing with JUNB and CTNNB1 mRNA, and to repress translation due to recruitment of the RACK repressor to the mRNA-lncRNA complex⁴⁸. The unique finding presented in this study, which was not reported before, is that RaPID purification co-precipitated the lncRNA substrates (Supplementary Data 4).

The finding that TblncRNA-23 regulates gene expression and is involved in the transition both from early to late PCF and from PCF to metacyclic forms suggests that ncRNAs play a major role in regulating the parasite developmental cycle within the insect host. Until the current study and a recent analysis of GRUMPY snoRNA²³ only RNA binding proteins were known to regulate transformation from one stage to the other^6,49. Interestingly, despite the high expression of metacyclic-specific genes as a result of TblncRNA-23 overexpression, the parasites did not transform to the metacyclic stage, most likely because they did not express RBP6 (Supplementary Fig. 10). It was previously shown that procyclin knockouts (which were generated by sequentially deleting pairs of procyclin genes and the intervening region) became progressively less efficient at colonizing the salivary glands and generating metacyclic forms⁵⁰. This was surprising at the time, because neither EP nor GPEET is expressed in the glands. These results can now be understood since each round of gene deletion removed a copy of TblncRNA-23 and the procyclin null mutant was also a TblncRNA-23 knockout. In conclusion, this study highlights the central roles of a lncRNA in the developmental cycle of T. brucei.

Methods

Cell growth and transfection

PCF T. brucei, strain 29-13⁵¹, which carries integrated genes for T7 polymerase and the tetracycline repressor, was grown in SDM-79 medium supplemented with 10% fetal calf serum, in the presence of 50 μg/ml hygromycin and 15 μg/ml G418 to generate the RNAi silenced cell lines. The TblncRNA-23 wild-type and mutants were overexpressed from plasmid pHD1700 (https://www.zmbh.uni-heidelberg.de/Clayton/vectors.html).

RNAi construct preparation

Stem-loop constructs were generated to silence TblncRNA-23 and PUF7 using primers listed in Supplementary Table 1, as described⁵¹. Stem-loop constructs were linearized by EcorV digestion and integrated into the non-transcribed rRNA spacer.

CRISPR-Cas9 knockout

For the inducible expression of SpCas9 in T. brucei, a plasmid (Cas9-2NLS-eYFP-BSD) was generated by cloning Streptococcus pyogenes Cas9 from plasmid Cas9-NLS-PPOTv4-PURO⁵² into plasmid 4296-SCD6-2NLS-eYFP-BSD (a gift from Dr Susanne Kramer, University of Wuerzburg, Germany). This plasmid was transfected into T. brucei procyclic 29/13 strain. TblncRNA-23 specific guide RNAs (gRNAs) targeting 5’ and 3’ regions of the gene were designed using the EuPaGDT database (http://grna.ctegd.uga.edu/). PCR product carrying T7 promoter and gRNA was prepared. To prepare a homologous DNA repair (HDR) template, PPOTv4 plasmid carrying coding sequences for tagRFPt and puromycin resistance was used as a template for PCR amplification. To increase the efficiency of HDR integration, long primers carrying 100nt complementarity sequences in both 5’ and 3’ directions were used. The primers used for gRNA synthesis and HDR template are listed in (Supplementary Table 1). SpCas9 expression was induced 12 h prior to transfection with PCR products of gRNAs and HDR. Transfected cells were selected using puromycin, single cells were cloned using Fluorescence-activated cell sorting (FACS), and KO cells were screened by Northern analysis.

Preparation of nuclear and whole cell extracts

T. brucei PCF (10⁸) were harvested and washed with phosphate-buffered saline. The cell pellet was resuspended in hypotonic buffer (10 mm HEPES, pH 7.9, 1.5 mm MgCl₂, 10 mm KCl, 0.5 mm dithiothreitol, 5 μg/ml leupeptin). For whole cell extract, 150 mM KCl salt was used; the cells were subjected to 20 strokes in a Dounce homogenizer in the presence of 0.1% Nonidet P-40. For nuclear preparation, the nuclei were pelleted at 10,000 × g, and proteins were extracted in 400 mM KCl.

Fractionation of RNPs on sucrose gradient

Whole cell extracts were prepared from 5 × 10⁹T. brucei PCF in a buffer containing 150 mM KCl, 20 mM Tris-pH 7.6, 10 mM MgCl₂, 0.5 M DTT and 0.1% NP-40. In addition, 1 μl leupeptin (10 μg/ml) protease inhibitor and 1 μl of RNasin (Thermo Scientific) were added to the lysate. Extracts were fractionated on a 10–30% sucrose gradient by centrifugation for 3 h at 210,000 g in a Beckman SW41 rotor at 4^oC. Gradient fractions were used for Western or Northern analyses.

Western analysis

Whole-cell lysates (10⁷ cells) were fractionated by SDS-PAGE, transferred to Protran membranes (Whatman), and reacted with the indicated antibodies. The bound antibodies were detected with goat anti-rabbit immunoglobulin G coupled to horseradish peroxidase and were visualized by ECL-enhanced chemiluminescence (Amersham Biosciences). The dilutions used for the antibodies are EP [Cedarlane (USA) 1:1000], GPEET (1:1000)⁴, PTB2 (1:10,000)⁵³, ZC3H41 (1:10,000)⁵⁴, GARP (1:10,000)⁵⁵, NRG1 (2:10,000)⁴², and BARP (2:10,000)⁴⁴. Quantitation of the signals was determined using ImageJ (https://imagej.nih.gov/ij/) software.

Northern analysis

Total RNA was prepared with TRIzol reagent (Sigma), and 20 μg/lane was fractionated on a 1.2% agarose, 2.2 m formaldehyde gel. After transfer, membranes were probed with anti-sense RNA probes. For analyzing small RNAs, total RNA was fractionated on a 6% polyacrylamide gel containing 7 m urea. The RNA was transferred to a nylon membrane (Hybond; Amersham Biosciences) and hybridized with anti-sense RNA probes. The antisense RNA probes were transcribed in vitro by T7 polymerase using a PCR product carrying the T7 promoter as a template. Quantitation of the signals was determined using ImageJ (https://imagej.nih.gov/ij/) software.

In situ hybridization

For high-resolution microscopy, T. brucei PCF were fixed on coverslips using 1.6% formaldehyde and permeabilized using Triton/Tween 20 (1:0.1%) in PBS. In situ hybridization with specific DIG-labeled RNA probes was performed as described⁵⁴. The nucleolus was localized using anti-NHP2 antibody. Images were acquired using a Leica SP8 confocal microscope equipped with a white light laser and gating.

RapID of TblncRNA-23

To identify proteins that associate with TblncRNA-23, a boxB sequence that binds the λ peptide⁵⁶ was introduced at position 133 with respect to the SL addition site using PCR with primers carrying the boxB sequence (listed in Supplementary Table 1). The TblncRNA-23 sequence tagged with boxB was cloned into the pHD1700 vector between the ApaI and MluI restriction sites, linearized with NotI, and integrated into the rRNA spacer in a cell line expressing the BirA-λ (biotin ligating enzyme-fused to the λ peptide) from the pPPOTv4 vector⁵⁷. The proteins biotinylated by BirA-λ were purified on streptavidin beads, as previously described⁵⁶ and subjected to tandem mass spectrometry, LC-MSMS, at the Smoler Proteomics Center, Technion, Israel. The mass spectrometry proteomics data was analyzed using the MaxQuant software version 2.1.1.0³⁸, and have been deposited to the ProteomeXchange Consortium via the PRIDE⁵⁸ partner repository with the dataset identifier PXD049995.

Poly-A RNA-seq analysis

The RNA-sequencing reads were aligned to the T. brucei genome (v5) using Smalt v_0.7.5 (http://www.sanger.ac.uk/resources/software/ SMALT/) with default parameters. To quantify read counts per gene, multiBamCov from bedtools (v2.30.0) was used to count aligned reads from BAM files. The gene count tables were used as input for the R-Bioconductor package, DESeq2 (v1.38.3)⁵⁹. The differential expression was assessed using the Wald test (default in DESeq2), and p-values were adjusted using the Benjamini-Hochberg (FDR) correction. mRNAs with an absolute log2 fold-change of >/=1 and FDR < 0.05 were considered differentially expressed. The volcano plot was generated using ggplot2 in R. To evaluate the significance of gene enrichment within list of genes a chi-square test was performed in R using the chisq.test function from https://www.R-project.org/. All sequencing data related to this study have been deposited in the NCBI BioProject database under the accession number PRJNA1066091.

Proteolysis and dimethyl labeling

Cells lysed using SDS buffer (5% SDS, 10 mM DTT, 100 mM Tris pH 8), sonicated twice (90%, 10-10 cycles, 5’), and centrifuged. The proteins in the solution were reduced (60 °C for 30 min), modified with 35.2 mM iodoacetamide in 100 mM ammonium bicarbonate (in dark, at room temperature for 30 min), and digested in 1.5 M Urea, 70 mM ABC with modified trypsin (Promega) at a 1:50 enzyme-to-substrate ratio, overnight at 37^oC. A second digestion was performed for 4 h. The resulting peptides were desalted using C18 Top tip columns (Glygen), dried, and re-suspended in 50 mM Hepes (pH 7.9). Labeling by dimethylation was performed as recently described (Rajan et al.⁵²) at the Smoler Proteomics Center, Technion, Israel, and raw data have been deposited to the ProteomeXchange Consortium via the PRIDE⁵⁸ partner repository with the dataset identifier PXD049995.

Mass spectrometry analysis of proteins

The peptides were resolved by reversed-phase chromatography on 0.075 × 180-mm fused silica capillaries (J&W) packed with Reprosil reversed phase material (Dr Maisch GmbH, Germany). The peptides were eluted with a linear 180 min gradient of 5 to 28%; a 15 min gradient of 28 to 95%; and 25 min with 95% acetonitrile and 0.1% formic acid in water, at a flow rate of 0.15 μl/min. Mass spectrometry was performed by Q-Exactive HFX mass spectrometer (Thermo) in positive mode using repetitively full MS scan followed by collision to induce dissociation (HCD) of the 30 most dominant ions selected from the first MS scan. The mass spectrometry data were analyzed using the Proteome Discoverer 1.4 (Thermo) software, searching against the T. brucei from the TriTrypDB v.35 database (https://tritrypdb.org). Results were filtered with rank 1 peptides and 1% false discovery rate. The ratios were normalized according to the protein’s median ratio. Perseus software (https://maxquant.net/perseus/) was used for statistical analysis of the data. Significant outliers relative to a given population were calculated using intensity-dependent calculation. The truncation was based on the Benjamini-Hochberg correction for multiple hypothesis testing (Significance B). For the combined analysis of different replicates, one-sample t-test was used to determine if the mean was significantly different from a fixed value (0).

RNA immunoprecipitation and qPCR for TblancRNA-23

Extracts from cells (10⁹) that express PUF7 N’PTP⁴² were prepared as previously described⁵⁴. RNA was extracted from the total cell extract, and the supernatant and the beads were subjected to RT-qPCR reactions in a Bio-Rad CFX96 thermal cycler. Relative normalized expression of target TBlncRNA-23 relative to total RNA and supernatant RNA were calculated using alpha tubulin mRNA as a reference gene according to the formula of ΔΔCq in CFX Maestro^TM Software (https://www.bio-rad.com/en-il/product/cfx-maestro-software-for-cfx-real-time-pcr-instruments).

Cells Flow cytometry analysis of cells expressing EP and GPEET

Cells (5 × 10⁵) were washed and blocked with 2% FBS, and incubated with anti-GPEET (2:1000) and anti-EP (1:10000) antibodies. After washing, cells were further incubated with Alexa Fluor 488-conjugated anti-rabbit IgG secondary antibody (1:1000; 2% FBS/PBS) for GPEET and with Cy5-conjugated anti-mouse IgG secondary antibody (1:1000; 2% FBS/PBS) for EP. The cells were washed and subjected to flow cytometry analysis in a BD LSR Fortessa instrument (BD Biosciences, San Jose, CA, United States). Forward scatter (FSC) and side scatter (SSC) were measured using a 488 nm laser. The 640 nm laser – 670/14 nm detector pair was used to determine the auto-fluorescence of cells. Post-acquisition analysis was done using FlowJo software (ver. 10.9) (Ashland, OR, United States).

Motility assay

Plates were prepared as described⁴. To inoculate the parasites, 5 μl of cell suspension (10⁶ or 2 × 10⁶ cells/ml) was spotted onto the agarose surface, and the plates were sealed with parafilm and incubated at 27 °C. Colony lift was performed as described⁴. The blots were reacted with antibodies to EP [Cedarlane (USA) 1:1000)] or GPEET (1:1000), and antibody binding was detected with IgG coupled to horseradish peroxidase and visualized by ECL.

Statistics and reproducibility

All experiments were repeated at least three times with biological replicates unless otherwise indicated. Poly(A) transcriptome, dimethyl labelled proteome, and RaPID (400 mM) were repeated two times. RaPID (150 mM) was repeated seven times, but only four experiments are presented. Statistical analysis and bar-graphs were prepared using Microsoft Excel and DESeq2 (v1.38.3).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The data supporting the findings of this study are available from the corresponding authors upon request. Source data are provided with this paper. The RNA sequencing data generated in this study have been deposited in the NCBI BioProject database under the accession number PRJNA1066091. The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE⁵⁸ partner repository with the dataset identifier PXD049995. For all main figures and supplemental figures Source data are provided with this paper.

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Acknowledgements

This work was supported by a grant from the Israel USA Binational Fund to S.M and C.T 20211103. S.M. holds the David and Inez Myers Chair in RNA silencing of diseases. We wish to thank the Smoler Proteomics Center, Technion, Israel, and Dr. Tamar Ziv for conducting and analyzing our proteome data.

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Authors and Affiliations

The Mina and Everard Goodman Faculty of Life Sciences and Advanced and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan, 52900, Israel
Beathrice Galili-Kostin, K. Shanmugha Rajan, Yuval Ida Ashkenazi, Almog Freedman, Tirza Doniger, Smadar Cohen-Chalamish, Hiba Waldman Ben-Asher, Ron Unger & Shulamit Michaeli
University of Bern, Bern, Switzerland
Isabel Roditi
Yale School of Public Health, Department of Epidemiology and Microbial Diseases, New Haven, CT, 06536, USA
Christian Tschudi

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Beathrice Galili-Kostin
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K. Shanmugha Rajan
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Yuval Ida Ashkenazi
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Contributions

S.M., I.R., C.T., conceptualization; B.K.G., K.S.R., Y.I.A., A.F., T.D., S.C.C., H.W.B. methodology; B.K.G., T.D., S.C.C., H.W.B. formal analysis; S.M., B.K.G., I.R., C.T., writing - original draft; S.M., I.R., C.T., R.U. supervision and project administration; S.M., C.T., R.U. funding acquisition.

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Correspondence to Shulamit Michaeli.

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Nature Communications thanks Keith Matthews, Lysangela Alves and Wei Hu, for their contribution to the peer review of this work. A peer review file is available.

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Galili-Kostin, B., Rajan, K.S., Ida Ashkenazi, Y. et al. TblncRNA-23, a long non-coding RNA transcribed by RNA polymerase I, regulates developmental changes in Trypanosoma brucei. Nat Commun 16, 3697 (2025). https://doi.org/10.1038/s41467-025-58979-w

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Received: 20 March 2024
Accepted: 08 April 2025
Published: 18 April 2025
Version of record: 18 April 2025
DOI: https://doi.org/10.1038/s41467-025-58979-w

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