1. Volume 28, Issue 2, Pages 174-188 (January 2014)
Functional Diversification of Dicer-like Proteins and Small RNAs Required for Genome Sculpting 
Pamela Y. Sandoval, Estienne C. Swart, Miroslav Arambasic, Mariusz Nowacki 
Developmental Cell 
Volume 28, Issue 2, Pages (January 2014)
DOI: /j.devcel
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2. Figure 1
Paramecium Dicer-like Proteins and the Effects of Their Silencing
(A) MrBayes phylogeny of Dicer (Dcr) and Dicer-like (Dcl) proteins. Posterior probabilities for each branch are shown, and the Dicer-like protein clade producing genome development-specific sRNAs is indicated in orange. A scale bar in expected substitutions per site is provided for branch length. Asterisks indicate the Dicer-like proteins characterized in this article. Pfam protein domains predicted using InterProScan (Goujon et al., 2010) HMMER3 ( searches of the Pfam database (Punta et al., 2012) are drawn below their protein (all to scale). Ciliate Dicer-like proteins only have detectable RNase III domain pairs. Note that the ResIII (Type III restriction enzyme) domain belongs to the same PFAM clan (CL0023) as the Helicase_C and DEAD domains. For Tetrahymena Dcr2 in the phylogeny, we used the RNase III domain region from Genbank accession BAD34723, and the domain image is from XP_ (these annotations conflict; i.e., BAD34723 is missing part of the ResIII helicase domain, and XP_ has a region between the two RNase III domains deleted).
(B) Gene expression profile of Dicer-like paralogs from vegetative culture (veg) and different stages of development. Gene expression levels in arbitrary units (in thousands) are from a previous study (Arnaiz et al., 2010). According to (Arnaiz et al., 2010), the five developmental stages following vegetative growth are: (1) cells undergoing meiosis, at the beginning of MAC fragmentation; (2) 50% of cells with fragmented old MAC (corresponding to our early time point); (3) significant proportion of cells with a visible new MAC; (4) late developmental stage where the majority of cells have a detectable new MAC (approximately corresponding to our late time point); and (5) population of cells 10 hr after stage 4.
(C) Localization of Dcl5-GFP in the developing new MAC. Fragments of the old MAC (DAPI-stained) do not appear to contain Dcl5-GFP.
See also Figure S1 and Supplemental Experimental Procedures.
Developmental Cell  , DOI: ( /j.devcel )
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3. Figure 2
Dicer-like Protein Gene Silencing Affects Paramecium Developmental sRNA Populations
(A) Radioactively 5′ end-labeled total RNA from control RNAi (Paramecium cells fed with E. coli producing dsRNAs corresponding to the plasmid L4440 with no sequence target in the Paramecium genome), DCL5, and DCL2/3 silenced cultures (Dicer-like silencing 1). E, M, and L indicate the early, middle, and late developmental stages (Control 1 from Experiment 1), respectively (panel C).
(B) Radioactively 5′ end-labeled total RNA from control, DCL5, DCL2, DCL3, and DCL2/3 silenced cultures (Dicer-like silencing 2); a schematic diagram illustrates the primary sRNAs visible in the gel image (compare to Figure 3). E and L indicate the early and late developmental stages (Control 2 from Experiment 2), respectively (Figure 2C).
(C) Distribution of cytological stages of developmental samples used in this study. A schematic representation of the fraction of cells scored in each sample is shown under the graph, with the graph colored according to the observed developmental stages. Bars represent the percentages of cells in the vegetative stage or during the following successive stages of MAC development: “Skein” indicates that cells with skein formation display parental MACs stretched to a characteristic convoluted structure; “Fragments” indicate that cells in this stage present fragmentation of the parental MAC with no evident new MAC; and “New MAC” indicates cells at later stages of development with fragmented MAC and a new developing MAC.
(D) Efficient silencing of Dicer-like transcripts was assayed by northern blot. Total RNA samples from the early (E) and late (L) developmental stages (Experiment 2) were hybridized with probes against the DCL2, DCL3, or DCL5 transcripts. The gel image was used to quantify the respective probe signal using ImageJ (Schneider et al., 2012). Background noise was subtracted, and the small subunit rRNA loading control was used for normalization (quantity is in arbitrary units).
(E) Total RNA treated with or without RNase A.
(F) Radioactively 5′ end-labeled total RNA from control and PGM silenced cells. M and L indicate the middle and late developmental stages for Control 2 (Dicer-like silencing 2) and PGM silenced cells (Experiment 3), respectively.
Developmental Cell  , DOI: ( /j.devcel )
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4. Figure 3
Genomic Distribution and Sequence Logos of Paramecium sRNAs from Control and Dicer-like Protein Gene Silencings
(A–E) Histograms of sRNAs binned according to length matching the MAC genome (green), IESs (red), and L4440 plasmid (purple; <50% GC) of the early and late developmental stages for control and silenced cells are shown on background histograms of total sRNAs (gray). See Experimental Procedures for details of mapping and normalization. Control, Control 2.
(F–J) Representative sequence logos of sRNAs matching IESs and the MAC genome. The proposed predominant class (or classes) of sRNAs underlying the sequence logos are indicated above each of the sequence logos. Only the first four bases (5′ end) and the last six bases (3′ end) of the sRNAs are shown, since the internal bases are typically close to the background base frequencies. Abundant MAC genome matching 27 nt sRNAs corresponding to a single locus are semitransparent, as they are not considered in this article. For the sake of clarity, we have not shown the 23 nt sRNA size class that predominantly comprises siRNAs, but this size class can be seen in the Supplemental Experimental Procedures.
(K) Sequence logos for 5′-non-U sRNAs from the early and late developmental stages (Control 1).
See also Figures S2 and S3.
Developmental Cell  , DOI: ( /j.devcel )
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5. Figure 4
IES Retention following Silencing of Genes Encoding Paramecium Dicer-like Proteins
(A and B) Retention of epi- and non-epiIESs was analyzed by PCR (A) for DCL2 and DCL3 silencing alone or combined (DCL2/3), and (B) for DCL5 silencing. Controls are cells as described in Figure 2 and Experimental Procedures. Names of the IESs are shown at the right of the IES schematics. IES retention percentages are estimated from Illumina DNA-seq data after silencing the Dicer-like genes (see Experimental Procedures).
(C) Genome-wide IES retention measured from Illumina sequencing of DNA from newly developed macronuclei for control, DCL2/3 cosilenced cells, and DCL5 silenced cells. For IESs with 0%–5% IES retention, the bars are truncated. The maximal values for these bars are 44,481, 39,953, and 38,295, for the control, DCL2/3 silenced, and DCL5 silenced cells, respectively.
(D) IES retention versus IES length (in base pairs) for DCL2/3 cosilenced and DCL5 silenced cells. Lines are the means of the IES retention calculated over 100 bp sliding windows with a step size of 1. To exclude potentially spuriously retained IESs, IESs with >5% retention in the control were filtered out. The y axis minimum value is set to −2%, in order to clearly show the low levels of IES retention in the DCL5 silencing. Anchois IESs are highlighted by large circles and are all longer than 1,000 bp.
(E) Mean iesRNA quantities from experimental cells (DCL2/3-KD or DCL5-KD) are normalized by the iesRNA quantities in control cells and plotted against percentage DCL2/3-KD IES retention. IES retention bins of 2% were used, and only bins containing ten or more IESs are shown, with the number of IESs in each bin shown in the lower subplot.
See also Table S1.
Developmental Cell  , DOI: ( /j.devcel )
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6. Figure 5
sRNA Coverage of a Select MIC Genome Region and Relative to IES Ends
(A and B) sRNA coverage of an arbitrary 15 kb MIC genome region with a high density of IESs (scaffold 76 of the reference Paramecium macronuclear genome assembly, corresponding to MAC genome positions 26,451–38,910) this locus was chosen for the purposes of visualization since IESs are small and most genomic regions have a relatively low density of IESs. Coverage plots were produced with a custom Python script, with each vertical line representing the per-base-pair sequence coverage. IESs are shown with boxes. The y axis scale is log2 [number of mapped reads/(library size per 108)]. Sequence data are from Control 2 (A) early and (B) late developmental stages respectively.
(C) sRNA densities (reads per IES bases) compared to IES length for all uniquely mapped 25 nt scnRNAs/iesRNAs and 26–29 nt sRNAs (iesRNAs) from Control 2.
(D–I) Pooled read coverage of mapped reads (from Experiment 2) surrounding TA repeats of IESs was calculated by binning the sRNA 5′ start (top of graphs) and 3′ end counts (bottom of graphs) normalized to the mean count of both strands for the 14 IES positions downstream of and including the TA repeat. sRNA coverage distributions are symmetrical with respect to the reverse complement of the downstream TA repeat. With the exception of (E), which only shows mapping of sRNAs to short IESs (26–36 bp long), sRNAs matching IESs >100 bp are shown. The source of sRNAs (control or silenced cells) and their specific sizes are indicated above each graph.
See also Figure S4.
Developmental Cell  , DOI: ( /j.devcel )
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7. Figure 6
Proposed Role of scnRNAs and iesRNAs in Paramecium DNA Elimination
The illustrated model is briefly described in the Discussion. Red arrows on either side of the IESs indicate a degenerate terminal-inverted repeat, starting/ending with TA. The remnant of excised IESs (a single TA) is indicated by a vertical red line. IES excision may be faciliated by scnRNAs or iesRNAs. A possible way to amplify iesRNAs, produced by Dcl5 in the new MAC (as seen in Figure 1C), from amplified IESs, is illustrated (lilac circuit).
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions