Splicing factor Spf30 assists exosome-mediated gene silencing in fission yeast
Authors: Sonia Dheur
Julie Drogat
Sylvie Genier
Pascal Bernard
Jean-Paul Javerzat
Journal: Molecular and Cellular Biology (2010)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Heterochromatin assembly; fission yeast; noncoding RNAs; RNA interference; exosome degradation pathways
Affiliations: CNRS, Institut de Biochimie et Génétique Cellulaires, UMR 5095, Bordeaux F-33077, France, Université Victor Segalen Bordeaux 2, Bordeaux F-33077, France

Abstract:  Heterochromatin assembly in fission yeast relies on the processing of cognate noncoding RNAs by both the RNA interference and the exosome degradation pathways. Recent evidence indicates that splicing factors facilitate the cotranscriptional processing of centromeric transcripts into small interfering RNAs (siRNAs). In contrast, how the exosome contributes to heterochromatin assembly and whether it also relies upon splicing factors were unknown. We provide here evidence that fission yeast Spf30 is a splicing factor involved in the exosome pathway of heterochromatin silencing. Spf30 and Dis3, the main exosome RNase, colocalize at centromeric heterochromatin and euchromatic genes. At the centromeres, Dis3 helps recruiting Spf30, whose deficiency phenocopies the dis3-54 mutant: heterochromatin is impaired, as evidenced by reduced silencing and the accumulation of polyadenylated centromeric transcripts, but the production of siRNAs appears to be unaffected. Consistent with a direct role, Spf30 binds centromeric transcripts and locates at the centromeres in an RNA-dependent manner. We propose that Spf30, bound to nascent centromeric transcripts, perhaps with other splicing factors, assists their processing by the exosome. Splicing factor intercession may thus be a common feature of gene silencing pathways. 

Retracted: No

PubPeer comments:
 May 2017 Correction, found here: https: mcb.asm.org content 37 11 e00115-17 It reads:  Fig. 4C: The panel for 37u00b0C *cen* transcript images was unintentionally replaced by a duplicate of the adjacent 32u00b0C panel. The correct 37u00b0C *cen* panel should appear as shown below. This correction has no consequence on the conclusion of the experiment and the validity of the study. We apologize for any confusion this mistake might have caused. Here is the original Figure 4C, in which not only the 32 and the 37u00b0C panels appear to be duplicates, but also the 25u00b0C panel. On the right a false-color version. ![file] All three replicates are shown as a continuous photo, making this in my professional opinion a category III duplication, in which parts within the same photo appear to be duplicated. Could the authors please explain how this duplication might have happened and why the 25u00b0C panel was not addressed in the correction? This paper was part of a structured search for image duplications in Molecular and Cellular Biology paper, which was published here: https: mcb.asm.org content 38 20 e00309-18.long , 
 Here is the original high-res (PMC) version of Figure 4C without any markings, to show that the image is presented as one photo with nine lanes. ![file] , 
 #2 Dear Elisabeth, Please find enclosed below (1) the jpeg images of the raw data Northern blots for cen and act1 RNA, we used to make figure 4C, along with the loading details (written below), and (2) the corrected figure 4C we sent to MCB. Hope this will clarify the situation and answer your questions. Sincerely, Pascal BERNARD ![file] ![file] ![file] Technical and loading details for Northen blots used to make Fig. 4C Cells were grown at 25u00b0C or 32u00b0C or shifted from 25u00b0C to 37u00b0C for 6h. Total RNA was extracted by hot phenol, 25 u00b5g was run in denaturing agarose gel, transferred to nytran SuPerCharge Whatman membrane, and blotted with cen or act1 probes. Lane 1 : wt at 25u00b0C Lane 2 : dcr1D at 25u00b0C Lane 3 : spf30-38 at 25u00b0C Lane 4 : dim1-35 at 25u00b0C Lane 5 : wt at 32u00b0C Lane 6 : dcr1D at 32u00b0C Lane 7 : spf30-38 at 32u00b0C Lane 8 : dim1-35 at 32u00b0C Lane 9 : wt at 25u00b0C Lane 10 : dcr1D at 25u00b0C Lane 11 : spf30-38 at 25u00b0C Lane 12 : dim1-35 at 25u00b0C Lane 13 : wt at 37u00b0C Lane 14 : dcr1D at 37u00b0C Lane 15 : spf30-38 at 37u00b0C Lane 16 : dim1-35 at 37u00b0C Samples collected at 25u00b0C have been loaded twice. , 
 Thank you, Dr. Bernard for posting these images here. The raw, uncropped blot you posted above looks different than the originally published figure. Lanes 1 2 3 look like the ones that were published to represent the 25, 32 and 37 degrees experiments, but the other lanes do not. The corrected figure, however, does seem to correspond to the original blot. Published figure correspond to lanes 1 2 3 1 2 3 1 2 3 of the uncropped blot Corrected figure correspond to lanes 1 2 3 5 6 7 13 14 15 of the uncropped blot Can you please clarify what happened that lanes 1 2 3 were repeated three times in the published blot? ,