The role of deadenylation in the degradation of unstable mRNAs in trypanosomes

Removal of the poly(A) tail is the first step in the degradation of many eukaryotic mRNAs. In metazoans and yeast, the Ccr4/Caf1/Not complex has the predominant deadenylase activity, while the Pan2/Pan3 complex may trim poly(A) tails to the correct size, or initiate deadenylation. In trypanosomes, turnover of several constitutively-expressed or long-lived mRNAs is not affected by depletion of the 5′–3′ exoribonuclease XRNA, but is almost completely inhibited by depletion of the deadenylase CAF1. In contrast, two highly unstable mRNAs, encoding EP procyclin and a phosphoglycerate kinase, PGKB, accumulate when XRNA levels are reduced. We here show that degradation of EP mRNA was partially inhibited after CAF1 depletion. RNAi-targeting trypanosome PAN2 had a mild effect on global deadenylation, and on degradation of a few mRNAs including EP. By amplifying and sequencing degradation intermediates, we demonstrated that a reduction in XRNA had no effect on degradation of a stable mRNA encoding a ribosomal protein, but caused accumulation of EP mRNA fragments that had lost substantial portions of the 5′ and 3′ ends. The results support a model in which trypanosome mRNAs can be degraded by at least two different, partially independent, cytoplasmic degradation pathways attacking both ends of the mRNA

The Trypanosome Pumilio Domain Protein PUF5

<div><p>PUF proteins are a conserved family of RNA binding proteins found in all eukaryotes examined so far. This study focussed on PUF5, one of 11 PUF family members encoded in the <i>Trypanosoma brucei</i> genome. Native PUF5 is present at less than 50000 molecules per cell in both bloodstream and procyclic form trypanosomes. C-terminally myc-tagged PUF5 was mainly found in the cytoplasm and could be cross-linked to RNA. PUF5 knockdown by RNA interference had no effect on the growth of bloodstream forms. Procyclic forms lacking PUF5 grew normally, but expression of PUF5 bearing a 21 kDa tandem affinity purification tag inhibited growth. Knockdown of <i>PUF5</i> did not have any effect on the ability of trypanosomes to differentiate from the mammalian to the insect form of the parasite.</p></div

PUF5-myc binds to RNA.

<p>A. Cells expressing myc-tagged PUF5-myc or UBP1-myc were UV-irradiated, protein was immunoprecipitated with anti-myc antibody, and the bound RNA was radioactively end-labelled. Samples were run on a denaturing SDS-PAGE and RNA detected by phosphorimaging. ‘*’ indicates the band corresponding to the ³²P labelled PUF5 bound RNAs while ‘**’ indicates the position of UBP1-bound RNAs. Wild type refers to the procyclic cell lines without any tagged protein. B. 10% of each sample was taken for western blot analysis using anti-myc antibody. ‘I’ refers to the input (cell lysate), ‘FT’ (flow through) refers to the unbound fraction and ‘E’ (eluate) refers to the bound fraction. Each sample represents approximately the same number of input trypanosomes.</p

PUF5-myc is in the cytoplasm.

<p>An aliquot of cells expressing C-terminally myc tagged PUF5 (see Fig. 1) was used to check the localization of the protein in procyclic trypanosomes. Wild-type cells were used as the negative control. BF refers to bright field. DAPI was used to stain the nucleus and kinetoplast.</p

<i>PUF5</i> knockdown does not affect differentiation of trypanosomes from the bloodstream to the procyclic form.

<p>Differentiation competent trypanosomes with <i>PUF5</i> RNAi were induced to differentiate into procyclic forms. This Figure shows a Western blot (top two panels) and a Northern blot (bottom two panels) for wild-type trypanosomes and two different RNAi clones, C1 and C2, after growth had resumed, but no difference was seen at any stage during the differentiation. The Western blot shows expression of EP procyclin and the Northern blot shows successful knockdown of <i>PUF5.</i> Ponceau-stained protein and methylene-blue stained rRNA serve as loading controls.</p

PUF5 is not essential for survival of PC trypanosomes.

<p>A. Cumulative growth curves of the bloodstream cells showing no difference in proliferation after <i>PUF5</i> RNAi or ectopic expression of C-terminally TAP-tagged PUF5. B. Western blot probed with anti-PUF5. C1 and C2 are different RNAi clones; their growth was indistinguishable. Ponceau S is the loading control. 5×10⁶cells loaded per lane. C. Northern blot for expression of <i>PUF5</i> RNA. Methylene-blue stained ribosomal RNA bands served as loading controls. D. Cumulative growth curves of procyclic cells ectopically expressing C-terminally myc tagged PUF5 or C-terminally TAP tagged PUF5. Results for procyclic <i>PUF5</i> double knockout cells (dKO) are also shown. E. Western blot to detect ectopic PUF5 expression, details as in (B). F. PCR amplification of a 443 bp fragment of the <i>PUF5</i> ORF in DNA from procyclic cells. A 708 bp fragment of <i>TbPUF2</i> ORF served as positive control. sKO: single knockout: dKO: double knock-out.</p

Endogenous PUF5 is not detected by a specific polyclonal antibody.

<p>Affinity purified anti-PUF5 antibody does not detect PUF5 in bloodstream-form (BS) and procyclic-form (PC) trypanosomes. Upper panel: From lanes 1 to 6, increasing amounts of recombinant protein were loaded. From lanes 7 to 11 and lanes 12 to 14, PC and BS cells were loaded, respectively. The very faint band that co-migrates with PUF5 in lanes 7, 8, 12 and 13 was background since it was also seen for the knockout (lane 9) and induced RNAi (lane 14). Lower panel: Ponceau S stain of the blot as a loading control.</p

author response efficacy of β lactam β lactamase inhibitor combination is linked to whib4 mediated changes in redox physiology of mycobacterium tuberculosis

n/

Efficacy of β-lactam/β-lactamase inhibitor combination is linked to WhiB4-mediated changes in redox physiology of Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) expresses a broad-spectrum β-lactamase (BlaC) that mediates resistance to one of the highly effective antibacterials, β-lactams. Nonetheless, β-lactams showed mycobactericidal activity in combination with β-lactamase inhibitor, clavulanate (Clav). However, the mechanistic aspects of how Mtb responds to β-lactams such as Amoxicillin in combination with Clav (referred as Augmentin [AG]) are not clear. Here, we identified cytoplasmic redox potential and intracellular redox sensor, WhiB4, as key determinants of mycobacterial resistance against AG. Using computer-based, biochemical, redox-biosensor, and genetic strategies, we uncovered a functional linkage between specific determinants of β-lactam resistance (e.g. β-lactamase) and redox potential in Mtb. We also describe the role of WhiB4 in coordinating the activity of β-lactamase in a redox-dependent manner to tolerate AG. Disruption of WhiB4 enhances AG tolerance, whereas overexpression potentiates AG activity against drug-resistant Mtb. Our findings suggest that AG can be exploited to diminish drug-resistance in Mtb through redox-based interventions

HSV-1-induced disruption of transcription termination resembles a cellular stress response but selectively increases chromatin accessibility downstream of genes.

Lytic herpes simplex virus 1 (HSV-1) infection triggers disruption of transcription termination (DoTT) of most cellular genes, resulting in extensive intergenic transcription. Similarly, cellular stress responses lead to gene-specific transcription downstream of genes (DoG). In this study, we performed a detailed comparison of DoTT/DoG transcription between HSV-1 infection, salt and heat stress in primary human fibroblasts using 4sU-seq and ATAC-seq. Although DoTT at late times of HSV-1 infection was substantially more prominent than DoG transcription in salt and heat stress, poly(A) read-through due to DoTT/DoG transcription and affected genes were significantly correlated between all three conditions, in particular at earlier times of infection. We speculate that HSV-1 either directly usurps a cellular stress response or disrupts the transcription termination machinery in other ways but with similar consequences. In contrast to previous reports, we found that inhibition of Ca2+ signaling by BAPTA-AM did not specifically inhibit DoG transcription but globally impaired transcription. Most importantly, HSV-1-induced DoTT, but not stress-induced DoG transcription, was accompanied by a strong increase in open chromatin downstream of the affected poly(A) sites. In its extent and kinetics, downstream open chromatin essentially matched the poly(A) read-through transcription. We show that this does not cause but rather requires DoTT as well as high levels of transcription into the genomic regions downstream of genes. This raises intriguing new questions regarding the role of histone repositioning in the wake of RNA Polymerase II passage downstream of impaired poly(A) site recognition