Invited Review Meeting Report: SMART Timing-Principles of Single Molecule Techniques Course at the University of Michigan 2014

ABSTRACT: Four days after the announcement of the 2014 Nobe

Photography-based taxonomy is inadequate, unnecessary, and potentially harmful for biological sciences

The question whether taxonomic descriptions naming new animal species without type specimen(s) deposited in collections should be accepted for publication by scientific journals and allowed by the Code has already been discussed in Zootaxa (Dubois & Nemésio 2007; Donegan 2008, 2009; Nemésio 2009a–b; Dubois 2009; Gentile & Snell 2009; Minelli 2009; Cianferoni & Bartolozzi 2016; Amorim et al. 2016). This question was again raised in a letter supported by 35 signatories published in the journal Nature (Pape et al. 2016) on 15 September 2016. On 25 September 2016, the following rebuttal (strictly limited to 300 words as per the editorial rules of Nature) was submitted to Nature, which on 18 October 2016 refused to publish it. As we think this problem is a very important one for zoological taxonomy, this text is published here exactly as submitted to Nature, followed by the list of the 493 taxonomists and collection-based researchers who signed it in the short time span from 20 September to 6 October 2016

Resolving Subcellular miRNA Trafficking and Turnover at Single-Molecule Resolution

Summary: Regulation of microRNA (miRNA) localization and stability is critical for their extensive cytoplasmic RNA silencing activity and emerging nuclear functions. Here, we have developed single-molecule fluorescence-based tools to assess the subcellular trafficking, integrity, and activity of miRNAs. We find that seed-matched RNA targets protect miRNAs against degradation and enhance their nuclear retention. While target-stabilized, functional, cytoplasmic miRNAs reside in high-molecular-weight complexes, nuclear miRNAs, as well as cytoplasmic miRNAs targeted by complementary anti-miRNAs, are sequestered stably within significantly lower-molecular-weight complexes and rendered repression incompetent. miRNA stability and activity depend on Argonaute protein abundance, whereas miRNA strand selection, unwinding, and nuclear retention depend on Argonaute identity. Taken together, our results show that miRNA degradation competes with Argonaute loading and target binding to control subcellular miRNA abundance for gene silencing surveillance. Probing single cells for miRNA activity, trafficking, and metabolism promises to facilitate screening for effective miRNA mimics and anti-miRNA drugs. : Pitchiaya et al. describe tools to interrogate gene-regulatory microRNAs inside living cells at single-molecule resolution. They find that the RNA silencing machinery and RNA targets mediate gene silencing surveillance by modulating the abundance and subcellular location of microRNAs. These findings and tools promise to facilitate single-cell screening of microRNA activity. Keywords: microRNA, Argonaute, mRNA targets, anti-miRs, correlative counting analysis, single-molecule microscop

The RNA binding protein RBM38 (RNPC1) regulates splicing during late erythroid differentiation.

Alternative pre-mRNA splicing is a prevalent mechanism in mammals that promotes proteomic diversity, including expression of cell-type specific protein isoforms. We characterized a role for RBM38 (RNPC1) in regulation of alternative splicing during late erythroid differentiation. We used an Affymetrix human exon junction (HJAY) splicing microarray to identify a panel of RBM38-regulated alternatively spliced transcripts. Using microarray databases, we noted high RBM38 expression levels in CD71(+) erythroid cells and thus chose to examine RBM38 expression during erythroid differentiation of human hematopoietic stem cells, detecting enhanced RBM38 expression during late erythroid differentiation. In differentiated erythroid cells, we validated a subset of RBM38-regulated splicing events and determined that RBM38 regulates activation of Protein 4.1R (EPB41) exon 16 during late erythroid differentiation. Using Epb41 minigenes, Rbm38 was found to be a robust activator of exon 16 splicing. To further address the mechanism of RBM38-regulated alternative splicing, a novel mammalian protein expression system, followed by SELEX-Seq, was used to identify a GU-rich RBM38 binding motif. Lastly, using a tethering assay, we determined that RBM38 can directly activate splicing when recruited to a downstream intron. Together, our data support the role of RBM38 in regulating alternative splicing during erythroid differentiation

Rbm38 activates Exon 16 in Epb41 minigenes.

<p>A) Schematic depicting a portion of the mouse protein 4.1R (Epb41) gene from exons 13 to 17 (Upper panel). In early erythroid differentiation, exons 14-16 are skipped, and in late erythroid differentiation exon 16 is included. Dashed lines indicate the position of exon 13 and 17 truncations in the minigenes described below. Closed and open arrows indicate primer binding sites that specifically amplify mRNA expressed from the minigenes and endogenous Epb41 locus, respectively. 4.1wt is a 1.2 kb minigene with indicated exonic and intronic sequences (Middle panel). Minigene 4.1Δhex lacks all three UGCAUG Rbfox2 binding motifs due to a 186-nt deletion within intron 16 (Lower panel). B) Co-transfection of minigenes and Empty vector (EV), Rbm38-FF, Rbfox2-FF, or both Rbm38-FF and Rbfox2-FF. <i>Upper </i><i>panel</i>, expression of Rbm38 and Rbfox2 promotes inclusion of Exon 16 in the 4.1wt minigene. When Rbm38 and Rbfox2 are expressed together, there is no further enhancement of exon 16 inclusion. <i>Lower </i><i>panel</i>, expression of Rbm38 promotes inclusion of Exon 16 in the 4.1 Δhex, while Rbfox2 has little effect. When Rbm38 and Rbfox2 are expressed together, activation of exon 16 splicing is similar to Rbm38 alone. We also noted a small band above the exon 16 inclusion product in Rbm38 lanes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078031#pone-0078031-g003" target="_blank">Figure 3B</a>, asterisk). This band was sequenced and found to contain exon 13, a retained intronic sequence downstream of exon 13, a region from intron 15, and exon 17. Exon 16 was not included. C) RT-PCR of endogenous EPB41 exon 16 inclusion in response to transfection of EV, Rbm38, Rbfox2, or both Rbm38 and Rbfox2. Percent exon inclusion is indicated below each lane. RT-PCR product sizes are provided to aid the reader in distinguishing minigene Epb41 from endogenous EPB41. D) Western blot analysis of Rbm38 and Rbfox2 protein expression levels. Actin was used as a loading control. </p

EPB41 exon 16 splicing increases during erythroid differentiation and exon 16 splicing is regulated by RBM38.

<p>A) Semi-quantiative RT-PCR detection of the activation of EPB41 exon 16 during late erythroid differentiation.).Average percentages of exon inclusion with standard deviations compiled from three experiments are indicated below a representative gel. For the middle time point, D7/8, average and standard deviation are calculated from one Day 7 and two Day 8 replicate samples. B) Semi-quantitative RT-PCR analysis of EPB41 exon 16 inclusion in MCF-7 cells after siRNA mediated knockdown of RBM38 from three biological replicate experiments. (C) RT-PCR analysis of EPB41 exon 16 splicing in response to knockdown of RBM38 in RL-7 cells RT-PCR products in RL-7 blood cell line were uncut or digested with BstE II (labeled as B) to detect presence of exon 14. D) Western blot detection of siRNA knockdown of RBM38 in RL-7 cells. Asterisk indicates a higher molecular weight RT-PCR product that is slightly enhanced in lanes with high RBM38 expression. </p

Mammalian expression and purification of Rbm38, followed by SELEX-Seq analysis to identify an RBM38 binding motif.

<p>A) Schematic of mammalian expressed Rbm38-FF-SBP purification steps. Mammalian 293T cells were transiently transfected for 48 h and protein was extracted using RIPA buffer. RIPA extract was added to Streptavidin resin and washed as indicated. The SBP tag was cleaved by incubation with TEV protease. Purified Rbm38-FF was used for SELEX-Seq. B) Map of FF-SPB tagged mammalian protein expression vector used in this study. Coomassie stain (C) and western blot (D) analysis of samples collected during purification of Rbm38-FF-SBP. E) Schematic for SELEX-Seq protocol. SELEX-Seq 7-mer motifs identified after two and three rounds of selection. </p

Direct tethering of Rbm38 to an intronic position downstream of a regulated exon activates splicing.

<p>A) Schematic of the PKC-40b-2xBoxB FGFR2 reporter minigene used in the lambda N-Box B tethering system. The minigene has a weak splice site, which promotes basal 40b exon skipping. The RNA sequence of Nut R Box B is provided in Materials and Methods B) Map of C- and N-terminal λN protein expression vectors. The amino acid sequence of lambda N peptide is provided in Materials and Methods C) Activation of 40b exon was examined by RT-PCR using RNA extracted from 293T cells transiently co-transfected for 48 h with expression vector: empty vector control (EV), Rbm38-FF, Rbm38-FF N-λN, or Rbm38-FF C-λN and minigene: no Box B insert or 2x Box B. Percent exon inclusion is provided below each lane. D) Western blot analysis of Flag tagged Rbm38 proteins used in (C). </p

RBM38 is expressed during late erythroid differentiation and RT-PCR analysis of a subset of RBM38-regulated microarray targets in erythroid differentiated cells.

<p>A) Western blot detection of RBM38 in erythroid differentiated CD34⁺ cells on days 2, 5, 7, and 11. (upper panel). Actin is shown as a loading control (lower panel). B) Quantitative RT-PCR of RBM38 mRNA expression levels during erythroid differentiation on days 3 to 13. C) Western blot detection of RBM38 in erythroid or granulocyte/monocyte differentiated CD34⁺ cells on days 7 and 10. SuperSignal West Femto Chemiluminescent ECL reagent was used to detect lower levels of RBM38 in early erythroid cells. D) Semi-quantitative RT-PCR analysis of RBM38 microarray targets ZDHHC18, ISOC2, and GUSB in erythroid differentiated cells. Average percentages of exon inclusion and standard deviations from three experiments are indicated below a representative gel. For the middle time point, D7/8, average and standard deviation are calculated from one Day 7 and two Day 8 replicate samples. </p

Validation of microarray predicted RBM38-regulated splicing events.

<p>A) Semi-quantitative RT-PCR analysis of HJAY microarray targets in MCF-7 cells. Average percentages of exon inclusion with standard deviations compiled from three experiments are indicated below a representative gel. . For AP1G2, only two replicates were included in analyses. B) Western blot detection of siRNA knockdown of RBM38 in MCF-7 cell line (upper panel). Actin is shown as a loading control (lower panel). C) Bar graph representing data from panel A. siControl and siRBM38 treated cells are shown in light and dark gray bars, respectively (<i>P</i>-values of ≤ 0.05 are annotated with an asterisk).</p