TBP and SNAP50 transcription factors bind specifically to the Pr77 promoter sequence from trypanosomatid non-LTR retrotransposons

Francisco Macías,Patricia E Carreira,M Carmen Thomas,Raquel Afonso-Lehmann

doi:10.1186/s13071-021-04803-5

Abstract

BackgroundTrypanosomatid genomes are colonized by active and inactive mobile DNA elements, such as LINE, SINE-like, SIDER and DIRE retrotransposons. These elements all share a 77-nucleotide-long sequence at their 5′ ends, known as Pr77, which activates transcription, thereby generating abundant unspliced and translatable transcripts. However, transcription factors that mediates this process have still not been reported.MethodsTATA-binding protein (TBP) and small nuclear RNA-activating protein 50 kDa (SNAP50) recombinant proteins and specific antibodies raised against them were generated. Protein capture assay, electrophoretic mobility-shift assays (EMSA) and EMSA competition assays carried out using these proteins and nuclear proteins of the parasite together to specific DNA sequences used as probes allowed detecting direct interaction of these transcription factors to Pr77 sequence.ResultsThis study identified TBP and SNAP50 as part of the DNA-protein complex formed by the Pr77 promoter sequence and nuclear proteins of Trypanosoma cruzi. TBP establishes direct and specific contact with the Pr77 sequence, where the DPE and DPE downstream regions are docking sites with preferential binding. TBP binds cooperatively (Hill coefficient = 1.67) to Pr77 and to both strands of the Pr77 sequence, while the conformation of this highly structured sequence is not involved in TBP binding. Direct binding of SNAP50 to the Pr77 sequence is weak and may be mediated by protein–protein interactions through other trypanosomatid nuclear proteins.ConclusionsIdentification of the transcription factors that mediate Pr77 transcription may help to elucidate how these retrotransposons are mobilized within the trypanosomatid genomes and their roles in gene regulation processes in this human parasite.Graphical

Highlights

Trypanosomatid genomes are colonized by active and inactive mobile DNA elements, such as Long interspersed nuclear element (LINE), Short interspersed nuclear element (SINE)-like, Short interspersed degenerated retroposons (SIDER) and Degener‐ ate ingi/L1Tc-related element (DIRE) retrotransposons
T. cruzi TATA-binding protein (TBP) and small nuclear RNA-activating protein 50 kDa (SNAP50) proteins bind to the Pr77 promoter sequence To investigate whether the TBP and SNAP50 proteins interact with the Pr77 DNA promoter sequence, an immunoassay was performed that included capture of parasite proteins that bind to Pr77 doublestranded DNA and subsequent protein identification by western blotting
The limited knowledge of the factors correlated with unusual modes of transcription in trypanosomatids and, in Trypanosome cruzi, highlights the need for studies to elucidate how transcriptional processes are mediated in these parasites

Summary

Introduction

Trypanosomatid genomes are colonized by active and inactive mobile DNA elements, such as LINE, SINE-like, SIDER and DIRE retrotransposons. These elements all share a 77-nucleotide-long sequence at their 5′ ends, known as Pr77, which activates transcription, thereby generating abundant unspliced and translatable transcripts. The genomes of these organisms are colonized by the nonLTR retrotransposons LINEs (long interspersed nuclear elements), such as L1Tc and ingi from T. cruzi and T. brucei, respectively; LINEs are autonomous retroelements that encode the enzymatic machinery that mediates their own transposition. The non-LTR retrotransposons SINEs (short interspersed nuclear elements), such as NARTc and RIME from T. cruzi and T. brucei, respectively, which colonize trypanosomatid genomes, are mobilized in trans by the LINE machinery, as they lack coding capacity. Trypanosomatid species contain degenerated retroelements, SIDERs (short interspersed degenerated retroposons) and DIREs (degenerate ingi/L1Tc-related elements), which have accumulated a large number of mutations that disable their mobilization capacity [7, 8]

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