MicroRNAs are exported from malignant cells in customized particles

MicroRNAs (miRNAs) are released from cells in association with proteins or microvesicles. We previously reported that malignant transformation changes the assortment of released miRNAs by affecting whether a particular miRNA species is released or retained by the cell. How this selectivity occurs is unclear. Here we report that selectively exported miRNAs, whose release is increased in malignant cells, are packaged in structures that are different from those that carry neutrally released miRNAs (n-miRNAs), whose release is not affected by malignancy. By separating breast cancer cell microvesicles, we find that selectively released miRNAs associate with exosomes and nucleosomes. However, n-miRNAs of breast cancer cells associate with unconventional exosomes, which are larger than conventional exosomes and enriched in CD44, a protein relevant to breast cancer metastasis. Based on their large size, we call these vesicles L-exosomes. Contrary to the distribution of miRNAs among different microvesicles of breast cancer cells, normal cells release all measured miRNAs in a single type of vesicle. Our results suggest that malignant transformation alters the pathways through which specific miRNAs are exported from cells. These changes in the particles and their miRNA cargo could be used to detect the presence of malignant cells in the body

Biogenesis of mammalian microRNAs by a non-canonical processing pathway

Canonical microRNA biogenesis requires the Microprocessor components, Drosha and DGCR8, to generate precursor-miRNA, and Dicer to form mature miRNA. The Microprocessor is not required for processing of some miRNAs, including mirtrons, in which spliceosome-excised introns are direct Dicer substrates. In this study, we examine the processing of putative human mirtrons and demonstrate that although some are splicing-dependent, as expected, the predicted mirtrons, miR-1225 and miR-1228, are produced in the absence of splicing. Remarkably, knockout cell lines and knockdown experiments demonstrated that biogenesis of these splicing-independent mirtron-like miRNAs, termed ‘simtrons’, does not require the canonical miRNA biogenesis components, DGCR8, Dicer, Exportin-5 or Argonaute 2. However, simtron biogenesis was reduced by expression of a dominant negative form of Drosha. Simtrons are bound by Drosha and processed in vitro in a Drosha-dependent manner. Both simtrons and mirtrons function in silencing of target transcripts and are found in the RISC complex as demonstrated by their interaction with Argonaute proteins. These findings reveal a non-canonical miRNA biogenesis pathway that can produce functional regulatory RNAs

Drosha Promotes Splicing of a Pre-microRNA-like Alternative Exon

<div><p>The ribonuclease III enzyme Drosha has a central role in the biogenesis of microRNA (miRNA) by binding and cleaving hairpin structures in primary RNA transcripts into precursor miRNAs (pre-miRNAs). Many miRNA genes are located within protein-coding host genes and cleaved by Drosha in a manner that is coincident with splicing of introns by the spliceosome. The close proximity of splicing and pre-miRNA biogenesis suggests a potential for co-regulation of miRNA and host gene expression, though this relationship is not completely understood. Here, we describe a cleavage-independent role for Drosha in the splicing of an exon that has a predicted hairpin structure resembling a Drosha substrate. We find that Drosha can cleave the alternatively spliced exon 5 of the <i>eIF4H</i> gene into a pre-miRNA both <i>in vitro</i> and in cells. However, the primary role of Drosha in <i>eIF4H</i> gene expression is to promote the splicing of exon 5. Drosha binds to the exon and enhances splicing in a manner that depends on RNA structure but not on cleavage by Drosha. We conclude that Drosha can function like a splicing enhancer and promote exon inclusion. Our results reveal a new mechanism of alternative splicing regulation involving a cleavage-independent role for Drosha in splicing.</p></div

Human Papillomavirus Type 16 Infection of Human Keratinocytes Requires Clathrin and Caveolin-1 and Is Brefeldin A Sensitive▿ †

Human papillomavirus type 16 (HPV16) has been identified as being the most common etiological agent leading to cervical cancer. Despite having a clear understanding of the role of HPV16 in oncogenesis, details of how HPV16 traffics during infection are poorly understood. HPV16 has been determined to enter via clathrin-mediated endocytosis, but the subsequent steps of HPV16 infection remain unclear. There is emerging evidence that several viruses take advantage of cross talk between routes of endocytosis. Specifically, JCV and bovine papillomavirus type 1 have been shown to enter cells by clathrin-dependent endocytosis and then require caveolin-1-mediated trafficking for infection. In this paper, we show that HPV16 is dependent on caveolin-1 after clathrin-mediated endocytosis. We provide evidence for the first time that HPV16 infection is dependent on trafficking to the endoplasmic reticulum (ER). This novel trafficking may explain the requirement for the caveolar pathway in HPV16 infection because clathrin-mediated endocytosis typically does not lead to the ER. Our data indicate that the infectious route for HPV16 following clathrin-mediated entry is caveolin-1 and COPI dependent. An understanding of the steps involved in HPV16 sorting and trafficking opens up the possibility of developing novel approaches to interfere with HPV16 infection and reduce the burden of papillomavirus diseases including cervical cancer

Drosha enhances exon 5 splicing.

<p>(A) Radiolabelled RT-PCR analysis of endogenous <i>eIF4H</i> splicing, and expression of <i>Drosha</i>, <i>DGCR8</i>, <i>DICER</i> and a loading control, <i>GAPDH</i>, in HEK-293T control cells or following transfection with Drosha, Dicer or TN Drosha. (B) Quantitation of exon 5 splicing. The graphs show the percent of exon 5 splicing [included/(included+skipped)*100]; TN Drosha n = 9; Drosha and Dicer n = 3. The lower graphs show quantitation of (C) <i>Drosha</i> (D) <i>DGCR8</i> (E) <i>TN Drosha</i> and (F) <i>DICER</i> abundance following overexpression, relative to <i>GAPDH</i>, n = 3. (G) Immunoblot of EIF4H protein isoform expression in the absence (control) or presence of TN Drosha expression in HEK-293T cells. (H) The graph shows the ratio of isoform expression (full length/skipped), n = 4. (I) The graph shows the overall protein expression of EIF4H [(Full Length+Skipped)/β-actin], n = 4. * p≤0.05, ** p≤0.005, *** p≤0.0005 indicate statistical significance by Student's t-test, All error bars represent SEM.</p

Drosha enhances exon 5 splicing directly.

<p>(A) The wild-type (WT) or Exon 5 ΔStructure minigenes templates that were used to generate RNA for the <i>in vitro</i> splicing assay are shown with the predicted exon 5 structures. Boxes represent exons and lines are introns. The red line in the ΔStructure mutant indicates the nucleotides that were mutated. (B) Immunoblot of FLAG-tagged GFP and Drosha that bound to anti-FLAG M2 magnetic beads (Sigma) and were used in the <i>in vitro</i> splicing assays. (C) Radiolabelled RT-PCR analysis of the RNA produced from <i>in vitro</i> splicing reactions of WT and Exon 5 ΔStructure RNA. RNA species (spliced and unspliced) are indicated. Splicing reactions were carried out in HeLa nuclear extracts in the absence of ATP (-ATP, lanes 1 and 4), or in the presence of ATP and supplemented with FLAG-tagged GFP (lanes 2 and 5), or Drosha (lanes 3 and 6). (D) Graph shows the percent of exon 5 splicing [spliced/(spliced+unspliced)*100].</p

<i>eIF4H</i> exon 5 co-immunoprecipitates with Drosha and TN Drosha.

<p>(A) An immunoblot of the FLAG-tagged proteins present in the input (lanes 1–3) and bound to the anti-FLAG M2 magnetic beads (Sigma) (bound, lanes 4–6). (B) Radiolabelled RT-PCR analysis of <i>eIF4H</i> unspliced RNA and mRNA in the input fraction (lanes 1–3) and bound to FLAG-tagged proteins (lanes 4–6) from HEK-293T cell lysates that were transiently transfected with <i>eIF4H</i> WT minigene and FLAG-tagged GFP (control), Drosha, or TN Drosha. <i>GAPDH</i> is a negative control. The top graph (<i>eIF4H</i> unspliced) shows the percent of unspliced RNA that was bound to the FLAG-tagged proteins, corrected for dilutions of Input relative to bound samples [bound/(input * dilution correction factor)]. The bottom graph shows the enrichment of exon 5 splicing in the bound fraction relative to input [(included/skipped)_bound/(included/skipped* dilution correction factor)_input]. Drosha values are an average of two independent experiments.</p

Exon 5 is cleaved from RNA <i>in vitro</i> and in cells.

<p>(A) RNA products isolated from a region of the gel from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004312#pgen-1004312-g002" target="_blank">Figure 2</a> corresponding to RNAs migrating between 30–80 nts in length were subjected to 5′ and 3′ RACE. Red arrows indicate 5′ and 3′ ends that were identified through sequencing of cleaved RNA products that represent the 5′ end and 3′ ends of putative 5p and 3p miRNAs, respectively. Black arrows indicate the 5′ and 3′ splice sites (ss). Bases outlined in red indicate a putative Dicer cleavage product as predicted by PhDCleav <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004312#pgen.1004312-Ahmed1" target="_blank">[39]</a>. The gray shading in the RNA structure represents exon 5. (B) Radiolabelled, stem-loop RT-PCR analysis of total cellular RNA, with (+) or without (−) reverse transcriptase, using stem-loop primers specific to the ends detected by sequencing of 3′ RACE products, or predicted by PHDCleav. SnoRNA65 (sno65) is a control, indicating the specific detection of RNA products. The stem-loop primer adds 38 nts to the amplicons, thus the 5p-miR and 3p/pre-miR are ∼36 nts and ∼27 nts, respectively, without the stem-loop. Molecular weight marker sizes are shown on the right. (C) Radiolabelled stem-loop RT-PCR analysis of exon 5 3p/pre-miR (n = 2) or (D) miR-126 (n = 3) in RNA isolated from HEK-293T control cells or cells transiently transfected with TN Drosha. Sno65 is a loading control. The graphs show the relative abundance of exon 5 3p/pre-miR and miR-126 RNA (miR/sno65). • indicates primer dimers. (E) <i>eIF4H</i> overall abundance as determined by Taqman qRT-PCR. Relative RNA quantity (RQ) was calculated using the ΔΔCt method where β-actin was the control, n = 6. In all cases, * indicates statistical significance determined by the Student's t-test where p≤0.05. Error bars represent standard error of the mean (SEM).</p