RNA turnover in Trypanosoma brucei.

Abstract

Regulation of variant surface glycoprotein (VSG) mRNA turnover in Trypanosoma brucei was studied in bloodstream forms, in procyclic cells, and during in vitro transformation of bloodstream forms to procycic cells by approach-to-equilibrium labeling and pulse-chase experiments. Upon initiation of transformation at 27°C in the presence of citrate-cis-aconitate, the half-life of VSG mRNA was reduced from 4.5 h in bloodstream forms to 1.2 h in transforming cells. Concomitantly, an approximately 25-fold decrease in the rate of transcription was observed, resulting in a 100-fold reduction in the steady-state level of de novo-synthesized VSG mRNA. This low level of expression was maintained for at least 7 h, finally decreasing to an undetectable level after 24 h. Transcription of the VSG gene in established procyclic cells was undetectable. For comparison, the turnover of polyadenylated and nonpolyadenylated RNA, ,-tubulin mRNA, and mini-exon-derived RNA (medRNA) was studied. For medRNA, no significant changes in the rate of transcription or stability were observed during differentiation. In contrast, while the rate of transcription of ,B-tubulin mRNA in in vitro-cultured bloodstream forms, transforming cells, and established procyclic cells was similar, the half life was four to five times longer in procyclic cells (tj/2, 7 h) than in cultured bloodstream forms (t1/2, 1.4 h) or transforming cells (t012, 1.7 h). Inhibition of protein synthesis in bloodstream forms at 37°C caused a dramatic 20-fold decrease in the rate of VSG mRNA synthesis and a 6-fold decrease in half-life to 45 min, while f8-tubulin mRNA was stabilized 2-to 3-fold and medRNA stability remained unaffected. It is postulated that triggering transformation or inhibiting protein synthesis induces changes in the abundance of the same regulatory molecules which effect the shutoff of VSG gene transcription in addition to shortening the half-life of VSG mRNA.The regulation of mRNA synthesis in the parasitic protozoan Trypanosoma brucei represents an interesting problem. First, throughout its complex life cycle in the mammalian host and the tsetse fly, this unicellular flagellate experiences dramatic changes in environment, to which it adapts by regulating the expression of numerous genes (42); second, it has the unique feature of discontinuous mRNA synthesis (4). In an as yet undefined mechanism, a 140-nucleotide (nt) transcript, designated mini-exon-derived RNA (medRNA), donates a 35-nt sequence located at its 5' end (mini-exon) to the 5' end of all mRNAs coding for proteins (8,9,12,21,26,44 (17,36,40). The mini-exon has been explicitly demonstrated at the 5' end of the VSG and tubulin mRNAs (3,17,36,41). medRNA is transcribed from mini-exon genes present in about 200 copies per nucleus. Most of these genes are clustered in the genome in tandemly linked 1.35-kilobase (kb) repeats (11,27 bloodstream forms to procyclic cells. In a second, related part, mRNA synthesis and decay in the absence of protein synthesis were investigated. MATERIALS AND METHODSTrypanos...

Transient activity assays of the Trypanosoma brucei variant surface glycoprotein gene promoter: control of gene expression at the posttranscriptional level.

“…The control of stability by signals in the 3' untranslated region of mRNAs is well documented (see references 16 and 37 for reviews). This could explain the observation that the VSG mRNA is much more stable than that of other ESAGs (9) and that its decay is strongly accelerated during differentiation into procyclic forms (12). Similarly, the levels of CAT activity resulting from the presence of the 3' untranslated regions from ESAG 7 and procyclin downstream of the CAT gene may reflect the relative stability of their respective mRNAs.…”

Trypanosoma brucei: posttranscriptional control of the variable surface glycoprotein gene expression site.

“…The transformation of bloodstream forms into procyclic forms can be mimicked in vitro (6,23). One of the main factors inducing this transformation is a reduction of temperature, and the first event observed is the arrest of transcription of the VSG gene (5,8). For several hours, this arrest is reversible upon restoration of the cultivation conditions for bloodstream forms, but after 24 h, transformation is irreversible (7,8).…”

SmD1 Is Required for Spliced Leader RNA Biogenesis

“…A key structural element of spliced leader RNA is the Smbinding site (11,16), RAU [4][5][6] GR, that is also present on many of the small nuclear RNAs (snRNAs) involved in splicing. The Sm-binding site interacts with a heptameric complex of Sm proteins (20).…”