Elements in the 3′ Untranslated Region of Procyclin mRNA Regulate Expression in Insect Forms of Trypanosoma brucei by Modulating RNA Stability and Translation

Abstract

Procyclins are the major surface glycoproteins of insect forms of Trypanosoma brucei. We have previously shown that a conserved 16-mer in the 3 untranslated region (UTR) of procyclin transcripts functions as a positive element in procyclic-form trypanosomes. A systematic analysis of the entire 297-base 3 UTR has now revealed additional elements which are involved in posttranscriptional regulation: a positive element which requires the first 40 bases of the 3 UTR and at least one negative element between nucleotides 101 and 173 (the LII domain). Deletion of either positive element resulted in a >8-fold reduction in the amount of protein but only an ϳ2-fold decrease in the steady-state level of mRNA, suggesting that regulation also occurred at the level of translation. In contrast, deletion of LII caused a threefold increase in the steady-state levels of both the mRNA and protein. LII-16-mer double deletions also gave high levels of expression, suggesting that the 16-mer functions as an antirepressor of the negative element rather than as an independent activator. All three elements have an effect on RNA turnover. When either positive element was deleted, the half-life (t 1/2 ) of the mRNA was reduced from ϳ50 min (the t 1/2 of the wild-type 3 UTR) to <15 min, whereas removal of the LII element resulted in an increased t 1/2 of ϳ100 min. We present a model of posttranscriptional regulation in which the negative domain is counteracted by two positive elements which shield it from nucleases and/or translational repressors.The differentiation of bloodstream forms of Trypanosoma brucei into procyclic forms which replicate in the tsetse fly midgut is marked by the synthesis of a new surface coat composed of procyclins (otherwise known as procyclic acidic repetitive proteins [PARPs]) and the shedding of the variant surface glycoprotein (VSG) coat, which covers the parasites in the mammalian host (31,42,54). It has been estimated that each cell is covered by approximately six million procyclin molecules (9) which are attached to the surface membrane by glycosylphosphatidylinositol (GPI) anchors (15, 16). The parasites divide by binary fission, with a population doubling time of ϳ9 to 10 h in culture, so there is a constant requirement for high levels of procyclin synthesis in order to maintain the density of the coat.Like the majority of genes in trypanosomatids, the procyclin genes form part of polycistronic transcription units (reviewed in reference 50). The trypanosome strain T. brucei 427 contains four procyclin expression sites which are located on separate chromosomes (43). Each expression site consists of tandemly linked procyclin genes (␣ and ␤), followed by a locus-specific procyclin-associated gene (PAG) (6, 28, 51). Two expression sites also contain an additional gene, GRESAG 2 (gene related to ESAG 2), which is very similar to a gene in the VSG expression site (4).When bloodstream form trypanosomes are triggered to differentiate into procyclic forms, there is a 5-to 10-fold increase in transcription initiatio...

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

“…1C) and appears to lack or to have a very short poly(A) tail because it was not recovered after an oligo(dT)-affinity column (Fig. 1C) and because we were unable to clone its 3Ј end by 3Ј RACE experiments by using an oligo(dT) 18 primer (results not shown). To further analyze whether the absence or reduced poly(A)-tail content of the TcUBP dicistron affects its mRNA stability, we measured its half-life by using RNA extracted from control and actinomycin D (ActD)-treated parasites (Fig.…”

“…1D). The half-life of this transcript is Ϸ5 h, indicating a normal or slower turnover rate compared with other short-lived mRNAs such as Amastin, EP1, Procyclin, and TcSMUG, the average half-life of which is Ϸ1 h (16)(17)(18)(19).…”

Expression of trans-Sialidase and 85-kDa Glycoprotein Genes in Trypanosoma cruzi Is Differentially Regulated at the Post-transcriptional Level by Labile Protein Factors

“…These elements have been identified in T. brucei procyclin mRNA as 16-mer stem-loop and 26-mer polypyrimidine tract sequences in the 3Ј-UTR, which, upon interaction with certain proteins, promote stabilization and translation of the target RNA, respectively (12,13). In T. cruzi, the stability of mRNA coding for reporter genes is also modulated by the 3Ј-UTR of some members of the 85-kDa glycoprotein family (6).…”

“…During processing, capped spliced leader sequence is added to the 5Ј-end, and a polyadenosine tail is added to the 3Ј-end of mRNA (10), probably ensuring mRNA stability and transport to the cytoplasm (11). In particular, correct splicing and polyadenylation, as well as the existence of specific 3Ј-untranslated portions of the transcribed genes, have been shown to promote either mRNA stability (12) or an increase in the translational efficiency (13). However, the nature of controlling factors and how environmental modifications induce differential expression remain obscure.…”

Presence of Multiple mRNA Cycling Sequence Element-binding Proteins in Crithidia fasciculata

“…Cisacting factors that affect mRNA stability have been identified within the 3Ј-untranslated region (UTR) 1 of specific transcripts. AU-rich cis-regulatory elements present in the 3Ј-UTR have been shown to regulate the stability of transcripts of procyclic acidic repetitive proteins (EP and GPEET) or procyclins (6,7) and the variable surface glycoprotein gene transcripts in Trypanosoma brucei (2,8) and those of mucin (9), amastin (10), and H2A histone (11) genes in Trypanosoma cruzi. Trans-acting factor, a developmentally regulated U-rich RNA-binding protein involved in selective mRNA destabilization, has recently been identified in T. cruzi (12).…”