Anna Jon Rasmussen's scientific contributions

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Publications (1)

FIGURE 1. Paralogs of the Paf1 complex are required for male fertility in D. melanogaster (A) Enrichment values and corresponding statistical significance levels for proteins associated with the Spermatocyte arrest (Sa; tTAF) bait protein as detected by proximity labeling mass-spectrometry. Blue highlights indicate basal transcription factors, including tTAF. Orange highlights indicate identified PAF1C-like proteins. (B) Schematic of the chromosomal position of PAF1C genes and the identified PAF1C gene paralogs. (C) Schematic of the aligned PAF1C and tPAF genes with the main protein domains as well as known interaction surfaces from PAF1C indicated. (D) mRNA levels of PAF1C and PAF1C paralog (tPAF) genes in selected adult tissues (RNAseq data from modENCODE, ref. 90 ), RPKM = Reads Per Kilobase per Million mapped reads. (E) Bar plot showing the mean embryo hatching rate in percent of eggs laid by control (w 1118 ) and tPAF mutant flies. Individual data points from biological replicates (n = 2) are shown as black dots.
FIGURE 3. tPAF forms a complex in vivo in spermatocytes that is distinct from canonical PAF1C (A) Western blot detection of endogenously tagged tPAF proteins using anti-FLAG antibodies. (B-C) Confocal images showing the GFP signal in testis tissue from endogenously tagged Paf1L tPAF (B) or Paf1 PAF1C (C) proteins with DAPI-stained DNA shown in magenta. (D-E) Enrichment values and corresponding statistical significance levels for proteins co-immunopurified (coIP) with endogenously tagged Ctr9L (D) or Paf1L (E) proteins as detected by mass-spectrometry (biological replicates, n, are indicated in F). (F) Bar plot showing the median quantified tPAF protein intensity of control and tPAF protein coIP-mass-spectrometry biological replicates. The individual replicate protein intensities are plotted with grey circles. (G-H) Predicted structure model of tPAF (G) and Drosophila PAF1C (H) complexes based on Alphafold2. Dashed lines indicate manually positioned structured domains. (I) Structural model of human PAF1C based on x-ray crystallography and cryo-electron microscopy related to the indicated PDB entries.
FIGURE 4. Spermatocyte-specific gene expression depends on tPAF (A) Confocal microscopy images showing the localization of GFP-tagged Cdc73L, Fibrillarin (Fib, anti-Fib immunoflourence staining), and chromatin visualized by H2Av-RFP. Dashed magenta line: nuclear border. Magenta arrowheads: position of major chromosome territories. (B) Bar plots showing the levels of tPAF gene mRNA based on bulk testis RNAseq from various tPAF mutants relative to control. (C) Enrichment values and corresponding statistical significance levels for mRNAs quantification based on bulk testis RNAseq in biological replicates of Leo1L (left panel) or Paf1L (right panel) tPAF mutants (n = 3) compared to control (n = 6) flies. Orange and blue arrows: the number of genes categorized as upregulated and downregulated, respectively. (D) Summary schematic of the identified interdependencies for nucleolar accumulation of tPAF proteins based on the data shown in Figure S16. (E) Enrichment values and corresponding statistical significance levels for mRNAs as quantified by RNAseq from FACS-sorted Cdc73L-GFP-positive spermatocytes in biological replicates of Leo1L (left panel) or Paf1L (right panel) tPAF mutant or control flies (n = 3). (F) Genome tracks showing RNA-seq coverage around the kl-5 gene for the indicated genotypes. Bin mean RPM: mean reads per million mapped reads (RPM) per genome region bin. (G) Bar plots showing the log2 fold change in mRNA levels of Y-linked genes, comparing tPAF mutant and control genotypes. Blue bars: mRNA level at least 50 % decreased in any tPAF mutant sample compared to control (Figure S14E). Green highlights: male fertility factor genes.
FIGURE 5. tPAF ensures efficient transcription termination at testis-specific genes (A) Genome tracks showing RNA-seq coverage around the unc79 gene for the indicated genotypes and sample type. The coverage signal is displayed as the log2 of mean reads per million mapped reads (RPM) per genome region bin. (B-C) Metagene analysis of RNAseq coverage fold change in Paf1L mutants relative to control samples. The standard error is plotted as a faded ribbon around the solid line showing the mean value at each genomic position. The plots compare coverage around bulk annotated genes ("other") to that of genes upregulated in tPAF mutants (B) or to that of genes showing increased downstream RNA-seq signal in tPAF mutants (C). (D) Violin plot of gene expression levels based on FlyAtlas2 RNAseq from the listed fly tissues. Expression is plotted separately for the gene categories established in (C). FPKM = Fragments Per Kilobase per Million mapped fragments. (E) Violin plot of spermatocyte RNAseq gene expression quantification of the gene categories established in (C). GeTMM = Gene length corrected trimmed mean of Mvalues. (F) Like (C-D) but here comparing read-in (RI) and read-through (RT) gene categories to the remaining ("other") annotated gene loci. (G) Box plots showing the distribution of change in gene expression values between mutant and control RNA-seq samples for the listed gene categories and mutant genotypes. The data in the tPAF mutant panels is the spermatocyte RNA-seq data. The data underlying the tTAF and tMAC panels originates from reference 49 .
A germline PAF1 paralog complex ensures cell type-specific gene expression
  • Preprint
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May 2024

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26 Reads

Astrid Pold Vilstrup

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Archica Gupta

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Anna Jon Rasmussen

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[...]

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Peter Andersen

Animal germline development and fertility rely on paralogs of general transcription factors that recruit RNA polymerase II to ensure cell type-specific gene expression. It remains unclear whether gene expression processes downstream of such paralog-based transcription is distinct from that of canonical RNA polymerase II genes. In Drosophila, the testis-specific TBP-associated factors (tTAFs) activate over a thousand spermatocyte-specific gene promoters to enable meiosis and germ cell differentiation. Here, we show that efficient termination of tTAF-activated transcription relies on testis-specific paralogs of canonical Polymerase Associated Factor 1 Complex (PAF1C) proteins, which form a testis-specific PAF1C (tPAF). Consequently, tPAF mutants cause aberrant expression of hundreds of downstream genes due to read-in transcription. Furthermore, tPAF facilitates expression of Y-linked male fertility factor genes, and thus broadly maintains spermatocyte-specific gene expression. Consistently, tPAF is required for the segregation of meiotic chromosomes and male fertility. Supported by comparative in vivo protein interaction assays, we provide a mechanistic model for the functional divergence of tPAF and PAF1C and for transcription termination as a developmentally regulated process required for cell type-specific gene expression.

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