Transcribed germline-limited coding sequences in Oxytricha trifallax

Abstract

The germline-soma divide is a fundamental distinction in developmental biology, and different genes are expressed in germline and somatic cells throughout metazoan life cycles. Ciliates, a group of microbial eukaryotes, exhibit germline-somatic nuclear dimorphism within a single cell with two different genomes. The ciliate Oxytricha trifallax undergoes massive RNA-guided DNA elimination and genome rearrangement to produce a new somatic macronucleus (MAC) from a copy of the germline micronucleus (MIC). This process eliminates noncoding DNA sequences that interrupt genes and also deletes hundreds of germline-limited open reading frames (ORFs) that are transcribed during genome rearrangement. Here, we update the set of transcribed germline-limited ORFs (TGLOs) in O. trifallax. We show that TGLOs tend to be expressed during nuclear development and then are absent from the somatic MAC. We also demonstrate that exposure to synthetic RNA can reprogram TGLO retention in the somatic MAC and that TGLO retention leads to transcription outside the normal developmental program. These data suggest that TGLOs represent a group of developmentally regulated protein-coding sequences whose gene expression is terminated by DNA elimination.

Keywords: DNA elimination; ciliate; genome rearrangement; germline; micronucleus; noncoding RNA.

Figure 1

Germline-limited ORFs are expressed during O. trifallax genome rearrangement. (A) Left: Steps for predicting transcribed germline-limited open reading frames (TGLOs) in the O. trifallax germline MIC genome. Center: Total number of computationally predicted candidates remaining after each pipeline step. Right: Total number of previously reported MIC-limited genes (Chen et al. 2014) remaining after each pipeline step. Yellow numbers (also leftmost numbers for the center and right columns) indicate high transcription TGLOs. Red numbers (also rightmost numbers for the center and right columns) indicate low transcription TGLOs. (B) EggNOG-mapper-predicted functions and conserved domains in high transcription TGLOs (upper yellow box) and low transcription TGLOs (lower red box). Blue text indicates that the associated TGLOs were validated by detection of peptides at a timepoint during rearrangement (Chen et al. 2014). (C) RNA sequencing reads from throughout the Oxytricha developmental life cycle were aligned to the MIC genome. Log₂-normalized RNA-seq read counts were calculated for high and low transcription TGLOs, as well as one thousand (randomly selected) somatic MAC-encoded genes across the O. trifallax developmental life cycle (hours labeled post mixing of compatible mating types: 0 hour, JRB310 and JRB510 cells mixed together; 12 hours, MIC meiosis; 18 hours, zygotic MAC formation; 24 hours, early rearrangement; 36 hours, mid-rearrangement; 48 hours, late rearrangement/disappearance of parental MAC; 60 hours, late rearrangement; 72 hours, end of rearrangement). Color scale refers to the log₂-normalized RNA expression. Rows are hierarchically clustered to clearly group TGLOs by their relative RNA expression at each time-point.

Figure 2

DNA from TGLO loci is eliminated from the developing MAC. (A) Log₂-normalized DNA copy number of high and low transcription TGLOs across the O. trifallax developmental life cycle based on mapping of whole-cell DNA sequencing reads to the MIC genome. Color scale refers to the log₂-normalized DNA copy number across each time course. Rows are hierarchically clustered to clearly group TGLOs by their relative DNA copy number at each time-point. (B) Nested telomere suppression PCR targeting the upstream telomere addition site of selected TGLOs in genomic DNA samples collected throughout the O. trifallax developmental life cycle. Detected PCR products correspond to a specific TGLO locus with an upstream telomere addition site that is not observed in asexually growing cells, and no assessed telomere-capped TGLOs are detectable after 74 hours.

Figure 3

Parental cells can carry a strain-specific germline-limited ORF. (A) Top: PCR targeting g111288 or Actin II using genomic DNA from F1 lines, parent lines, and other mutant F1 lines used in this study. Bottom: Genome track showing the approximate location of g111288 PCR primers. Yellow: g111288, light blue: flanking MDSs. (B) The germline genome locus containing g111288 with mapped F1 reads from a pool of asexually growing F1 cells. Yellow: g111288, light blue: MDSs, dark blue: assembled g111288 MDSs from pooled F1 reads, gray triangles: observed telomere addition sites. (C) The germline genome locus (bottom) containing g111288 (yellow) and strain-specific MDSs (dark blue) with mapped RNA-seq coverage (black) from several time-points during asexual growth (starved or encysted cells) and hours post mixing of mating types during the sexual life cycle. (D) Top: Copy number relative to mitochondrial rDNA based on qPCR targeting several amplicons on the g111288 nanochromosome, an IES within the corresponding germline locus, and two unrelated somatic loci. Bottom: The germline genome locus containing g111288 with qPCR primer locations indicated. Yellow: g111288, light blue: MDSs, dark blue: assembled g111288 MDSs from pooled F1 reads, black arrows: qPCR primers. (E) Top: Southern blot of parental and F1 MAC DNA detected using an MDS-MDS junction spanning DNA probe. Bottom: MIC genome track showing the portions of MDSs 1 and 2 detected. (F) Top: RT-PCR targeting g111288 or Actin II using RNA from the same cell lines as in (A). Bottom: Genome track showing the approximate location of g111288 RT-PCR primers. Yellow: g111288, light blue: MDSs.

Figure 4

TGLO loci have few Otiwi-1 piRNAs and template RNAs. (A) Distribution of normalized mapping quality-filtered Otiwi-1 piRNA read counts (Fang et al. 2012) mapped to high and low transcription TGLOs compared to MDSs. Read counts were normalized to reads per kilobase million (RPKM). Brackets and asterisks indicate statistically significant differences between distributions. Statistical significance was assessed using the nonparametric Kolmogorov–Smirnov (KS) test, and P < 0.05 was considered statistically significant. (B) The germline genome locus containing the strain-specific TGLO g111288 (yellow), MDSs (blue), and mapping of Otiwi-1-associated piRNAs (gray) from several time-points during rearrangement onto MDS 1-7 of g111288 and the flanking MDSs for other loci. (C) Distribution of normalized mapping quality-filtered template RNA read counts (Lindblad et al. 2017) mapped to high and low transcription TGLOs compared to MDSs. Read counts were normalized to RPKM. Brackets and asterisks indicate statistically significant differences between distributions. Statistical significance was assessed using the nonparametric KS test, and P < 0.05 was considered statistically significant. (D) The germline genome locus containing the strain-specific TGLO g111288 (yellow), MDSs (blue), and mapped template RNA coverage (gray) from several time-points during rearrangement.

Figure 5

RNA injection programs heritable TGLO retention. (A) Synthetic RNA injection scheme to program the retention of a TGLO (yellow) in an IES between two MDSs (blue blocks). Possible products can include telomere-capped (black blocks) nanochromosomes with the entire IES plus TGLO flanked by the MDSs of the wild-type flanking locus. (B) The germline genome loci containing the programmed retention candidate TGLOs g104149 and g67186 (yellow), MDSs (blue), and mapped piRNA or template RNA coverage (gray) from several time-points during rearrangement. (C) Top: Cell culture PCR targeting the IES containing g104149 from cell lines derived from single RNA injected mating pairs. Middle: The expected retention product containing g104149 with PCR primer locations. Yellow: g104149, light blue: MDSs, black arrows: PCR primers. Bottom: Sanger sequencing chromatograms from PCR reactions in (B) aligned to the expected retention product containing g104149 (yellow). (D) Top: Cell culture PCR targeting the IES containing the predicted Histone 2B TGLO g67186 from cell lines derived from single RNA injected mating pairs. Middle: The expected retention product containing g67186 with PCR primer locations. Yellow: g67186, light blue: MDSs, black arrows: PCR primers. Bottom: Sanger sequencing chromatograms from PCR reactions aligned to the expected retention product containing g67186 (yellow). (E) Top: PCR targeting the IES containing g104149 using genomic DNA from parental cells, F1 retention cells, F1 retention cells backcrossed to parental cells, and unmanipulated F1 lines. Bottom: The expected retention product containing g104149 with PCR primers. Yellow: g104149, light blue: MDSs, black arrows: PCR primers. (F) Top: PCR targeting the IES containing the predicted histone 2B TGLO g67186 using genomic DNA from parental cells, F1 retention cells, F1 retention cells backcrossed to parental cells, and unmanipulated F1 lines. Bottom: The expected retention product containing g67186 with PCR primers. Yellow: g67186, light blue: MDSs, black arrows: PCR primers.

Figure 6

Retained TGLOs are misexpressed during asexual life cycle. (A) Possible transcription start sites (black arrows) on a hypothetical rearranged somatic nanochromosome after RNA injection to somatically retain TGLOs (yellow). Green: target transcript deriving from TGLO’s putative upstream regulatory sequence. Blue blocks: MDSs. Black blocks: Telomeres. (B) Top: Germline genome locus containing g104149 (yellow) and gene-specific 5’ RACE primers used to amplify transcription start site. Bottom: 5’ RACE products targeting the g104149 or Actin II transcription start site in RNA from F1 cells from TGLO retention engineered line # 4, parental cells (JRB310 and JRB510), and late-rearrangement mated cells (WT 48 hours). TdT: terminal transferase. (C) Top: Germline genome locus containing g67186 (yellow) and gene-specific 5’ RACE primers used to amplify transcription start site. 5’ RACE products targeting the g67186 or Actin II transcription start site in RNA from F1 TGLO retention engineered line # 3 and #5, parental cells (JRB310 and JRB510), and late-rearrangement mated cells (WT 48 hours). TdT: terminal transferase. (D) g104149 or Actin II RNA transcript levels based on qRT-PCR relative to mitochondrial rRNA. Error bars: standard deviation of three biological replicates. (E) g67186 or Actin II RNA transcript levels based on qRT-PCR relative to mitochondrial rRNA. Error bars: standard deviation of three biological replicates.