mRNA Fragments Research Articles

Selective ribosome profiling to study interactions of translating ribosomes in yeast.

A number of enzymes, targeting factors and chaperones engage ribosomes to support fundamental steps of nascent protein maturation, including enzymatic processing, membrane targeting and co-translational folding. The selective ribosome profiling (SeRP) method is a new tool for studying the co-translational activity of maturation factors that provides proteome-wide information on a factor's nascent interactome, the onset and duration of binding and the mechanisms controlling factor engagement. SeRP is based on the combination of two ribosome-profiling (RP) experiments, sequencing the ribosome-protected mRNA fragments from all ribosomes (total translatome) and the ribosome subpopulation engaged by the factor of interest (factor-bound translatome). We provide a detailed SeRP protocol, exemplified for the yeast Hsp70 chaperone Ssb (stress 70 B), for studying factor interactions with nascent proteins that is readily adaptable to identifying nascent interactomes of other co-translationally acting eukaryotic factors. The protocol provides general guidance for experimental design and optimization, as well as detailed instructions for cell growth and harvest, the isolation of (factor-engaged) monosomes, the generation of a cDNA library and data analysis. Experience in biochemistry and RNA handling, as well as basic programing knowledge, is necessary to perform SeRP. Execution of a SeRP experiment takes 8-10 working days, and initial data analysis can be completed within 1-2 d. This protocol is an extension of the originally developed protocol describing SeRP in bacteria.

Polynucleotide phosphorylase promotes the stability and function of Hfq-binding sRNAs by degrading target mRNA-derived fragments.

In many Gram-negative and some Gram-positive bacteria, small regulatory RNAs (sRNAs) that bind the RNA chaperone Hfq have a pivotal role in modulating virulence, stress responses, metabolism and biofilm formation. These sRNAs recognize transcripts through base-pairing, and sRNA–mRNA annealing consequently alters the translation and/or stability of transcripts leading to changes in gene expression. We have previously found that the highly conserved 3′-to-5′ exoribonuclease polynucleotide phosphorylase (PNPase) has an indispensable role in paradoxically stabilizing Hfq-bound sRNAs and promoting their function in gene regulation in Escherichia coli. Here, we report that PNPase contributes to the degradation of specific short mRNA fragments, the majority of which bind Hfq and are derived from targets of sRNAs. Specifically, we found that these mRNA-derived fragments accumulate in the absence of PNPase or its exoribonuclease activity and interact with PNPase. Additionally, we show that mutations in hfq or in the seed pairing region of some sRNAs eliminated the requirement of PNPase for their stability. Altogether, our results are consistent with a model that PNPase degrades mRNA-derived fragments that could otherwise deplete cells of Hfq-binding sRNAs through pairing-mediated decay.

Monitoring Cell-Type-Specific Gene Expression Using Ribosome Profiling In Vivo During Cardiac Hemodynamic Stress.

Gene expression profiles have been mainly determined by analysis of transcript abundance. However, these analyses cannot capture posttranscriptional gene expression control at the level of translation, which is a key step in the regulation of gene expression, as evidenced by the fact that transcript levels often poorly correlate with protein levels. Furthermore, genome-wide transcript profiling of distinct cell types is challenging due to the fact that lysates from tissues always represent a mixture of cells. This study aimed to develop a new experimental method that overcomes both limitations and to apply this method to perform a genome-wide analysis of gene expression on the translational level in response to pressure overload. By combining ribosome profiling (Ribo-seq) with a ribosome-tagging approach (Ribo-tag), it was possible to determine the translated transcriptome in specific cell types from the heart. After pressure overload, we monitored the cardiac myocyte translatome by purifying tagged cardiac myocyte ribosomes from cardiac lysates and subjecting the ribosome-protected mRNA fragments to deep sequencing. We identified subsets of mRNAs that are regulated at the translational level and found that translational control determines early changes in gene expression in response to cardiac stress in cardiac myocytes. Translationally controlled transcripts are associated with specific biological processes related to translation, protein quality control, and metabolism. Mechanistically, Ribo-seq allowed for the identification of upstream open reading frames in transcripts, which we predict to be important regulators of translation. This method has the potential to (1) provide a new tool for studying cell-specific gene expression at the level of translation in tissues, (2) reveal new therapeutic targets to prevent cellular remodeling, and (3) trigger follow-up studies that address both, the molecular mechanisms involved in the posttranscriptional control of gene expression in cardiac cells, and the protective functions of proteins expressed in response to cellular stress.

The Clinical Potential of Oligonucleotide Therapeutics against Pancreatic Cancer.

Although many diagnostic and therapeutic modalities for pancreatic cancer have been proposed, an urgent need for improved therapeutic strategies remains. Oligonucleotide therapeutics, such as those based on antisense RNAs, small interfering RNA (siRNA), microRNA (miRNA), aptamers, and decoys, are promising agents against pancreatic cancer, because they can identify a specific mRNA fragment of a given sequence or protein, and interfere with gene expression as molecular-targeted agents. Within the past 25 years, the diversity and feasibility of these drugs as diagnostic or therapeutic tools have dramatically increased. Several clinical and preclinical studies of oligonucleotides have been conducted for patients with pancreatic cancer. To support the discovery of effective diagnostic or therapeutic options using oligonucleotide-based strategies, in the absence of satisfactory therapies for long-term survival and the increasing trend of diseases, we summarize the current clinical trials of oligonucleotide therapeutics for pancreatic cancer patients, with underlying preclinical and scientific data, and focus on the possibility of oligonucleotides for targeting pancreatic cancer in clinical implications.

Phospho‐RNA‐seq: a modified small RNA‐seq method that reveals circulating mRNA and lncRNA fragments as potential biomarkers in human plasma

Extracellular RNAs (exRNAs) in biofluids have attracted great interest as potential biomarkers. Although extracellular microRNAs in blood plasma are extensively characterized, extracellular messenger RNA (mRNA) and long non‐coding RNA (lncRNA) studies are limited. We report that plasma contains fragmented mRNAs and lncRNAs that are missed by standard small RNA‐seq protocols due to lack of 5′ phosphate or presence of 3′ phosphate. These fragments were revealed using a modified protocol (“phospho‐RNA‐seq”) incorporating RNA treatment with T4‐polynucleotide kinase, which we compared with standard small RNA‐seq for sequencing synthetic RNAs with varied 5′ and 3′ ends, as well as human plasma exRNA. Analyzing phospho‐RNA‐seq data using a custom, high‐stringency bioinformatic pipeline, we identified mRNA/lncRNA transcriptome fingerprints in plasma, including tissue‐specific gene sets. In a longitudinal study of hematopoietic stem cell transplant patients, bone marrow‐ and liver‐enriched exRNA genes were tracked with bone marrow recovery and liver injury, respectively, providing proof‐of‐concept validation as a biomarker approach. By enabling access to an unexplored realm of mRNA and lncRNA fragments, phospho‐RNA‐seq opens up new possibilities for plasma transcriptomic biomarker development.

Identifying A- and P-site locations on ribosome-protected mRNA fragments using Integer Programming

Identifying the A- and P-site locations on ribosome-protected mRNA fragments from Ribo-Seq experiments is a fundamental step in the quantitative analysis of transcriptome-wide translation properties at the codon level. Many analyses of Ribo-Seq data have utilized heuristic approaches applied to a narrow range of fragment sizes to identify the A-site. In this study, we use Integer Programming to identify the A-site by maximizing an objective function that reflects the fact that the ribosome’s A-site on ribosome-protected fragments must reside between the second and stop codons of an mRNA. This identifies the A-site location as a function of the fragment’s size and its 5′ end reading frame in Ribo-Seq data generated from S. cerevisiae and mouse embryonic stem cells. The correctness of the identified A-site locations is demonstrated by showing that this method, as compared to others, yields the largest ribosome density at established stalling sites. By providing greater accuracy and utilization of a wider range of fragment sizes, our approach increases the signal-to-noise ratio of underlying biological signals associated with translation elongation at the codon length scale.

SUN-030 Expression Profiles of Short RNAs in Mouse Serum Employing Endocrine Organs as Controls

Over 90% of the human genome expresses many different forms of non-protein-coding (nc) RNAs in addition to mRNAs. They are categorized into short and long ncRNAs. Although their overall functions and biological importance are still under investigation, they appear to be essential for supporting complex human activities mainly by regulating genome transcription and/or protein translation. It is known that some short RNAs are identified in several body fluids of humans, including serum, urine and saliva. Biological and/or pathological roles of serum micro (mi) RNAs have recently been reported, whereas little is known about other short non-coding (snc) RNAs circulating in serum. Here we present a comprehensive analysis on the short RNAs present in mouse serum using the RNA-sequencing by employing two endocrine organs (adrenal gland and ovary) and liver as controls. We found that serum has numerous short RNA reads and most of them (~90%) are unable to be mapped to the reference genome. Among the mapped reads, short RNAs expressed from the intergenic area (intergenic sncRNAs) represent the majority (97.7%) of short RNAs appeared in mouse serum. These intergenic sncRNAs are mapped to the intergenic area located closely to the nearby protein-coding genes (~20 kb distance), indicating their potential regulatory functions on the transcription of these coding genes. Most of the serum intergenic sncRNAs are distinct from those identified in the three control organs, whereas the intergenic sncRNAs found in the control organs highly overlap with each other. Among known sncRNAs, fragments of ribosomal (r) RNAs (72.0%), transfer (t) RNAs (18.1%), piwi-interacting (pi) RNAs (4.01%) and micro (mi) RNAs (3.96%) are most abundant, whereas small nuclear (sn) RNAs (1.46%) and small nucleolar (sno) RNAs (0.44%) are found as minor fractions. Very few short RNAs derived from mRNAs or long non-coding RNAs are also identified in mouse serum. Interestingly, many of the mRNA fragments identified in serum are derived from the genes associated with the transmembrane signaling pathways in the Ingenuity® Pathway Analysis, suggesting easy liberation of these mRNA fragments from respective cells possibly due to their local translation as reported in neuronal cells. Taken together, our findings indicate that sncRNAs, particularly intergenic sncRNAs, rRNA fragments and tRNAs, are abundant in mouse serum. These results suggest in humans that they may have important biological actions and/or potential clinical implications as valuable clinical markers/therapeutic targets in addition to miRNAs.

Ribosome profiling as an innovative tool to study the process of protein synthesis

The ribosome profiling approach (Ribo-seq) is currently the most effective method to study the protein synthesis in vivo. This technique relies on sequencing of ribosome protected mRNA fragments (so-called ribosomal footprints) allowing to indicate the exact positions of ribosomes on transcripts. Advanced bioinformatic analysis of Ribo-seq data enables selection of ribosomal footprints originated from translating ribosomes, providing information about authentic translational status of mRNA. Here, authors present principles of ribosome profiling method with special attention to basic experimental and bioinformatics strategies important for obtaining satisfactory results. Review includes examples of applications of ribosome profiling technique in various biological systems, giving the significant insight into the translation process and its regulation.

Towards DNA Nanomachines for Cancer Treatment: Achieving Selective and Efficient Cleavage of Folded RNA.

Despite decades of effort, gene therapy (GT) has failed to deliver clinically significant anticancer treatment, owing in part to low selectivity, low efficiency, and poor accessibility of folded RNA targets. Herein, we propose to solve these common problems of GT agents by using a DNA nanotechnology approach. We designed a deoxyribozyme-based DNA machine that can i) recognize the sequence of a cancer biomarker with high selectivity, ii) tightly bind a structured fragment of a housekeeping gene mRNA, and iii) cleave it with efficiency greater than that of a traditional DZ-based cleaving agent. An important advantage of the DNA nanomachine over other gene therapy approaches (antisense, siRNA, and CRISPR/cas) is its ability to cleave a housekeeping gene mRNA after being activated by a cancer marker RNA, which can potentially increase the efficiency of anticancer gene therapy. The DNA machine could become a prototype platform for a new type of anticancer GT agent.

Towards DNA Nanomachines for Cancer Treatment: Achieving Selective and Efficient Cleavage of Folded RNA

AbstractDespite decades of effort, gene therapy (GT) has failed to deliver clinically significant anticancer treatment, owing in part to low selectivity, low efficiency, and poor accessibility of folded RNA targets. Herein, we propose to solve these common problems of GT agents by using a DNA nanotechnology approach. We designed a deoxyribozyme‐based DNA machine that can i) recognize the sequence of a cancer biomarker with high selectivity, ii) tightly bind a structured fragment of a housekeeping gene mRNA, and iii) cleave it with efficiency greater than that of a traditional DZ‐based cleaving agent. An important advantage of the DNA nanomachine over other gene therapy approaches (antisense, siRNA, and CRISPR/cas) is its ability to cleave a housekeeping gene mRNA after being activated by a cancer marker RNA, which can potentially increase the efficiency of anticancer gene therapy. The DNA machine could become a prototype platform for a new type of anticancer GT agent.

Hantavirus RdRp Requires a Host Cell Factor for Cap Snatching.

The hantavirus RNA-dependent RNA polymerase (RdRp) snatches 5' capped mRNA fragments from the host cell transcripts and uses them as primers to initiate transcription and replication of the viral genome in the cytoplasm of infected cells. Hantavirus nucleocapsid protein (N protein) binds to the 5' caps of host cell mRNA and protects them from the attack of cellular decapping machinery. N protein rescues long capped mRNA fragments in cellular P bodies that are later processed by an unknown mechanism to generate 10- to 14-nucleotide-long capped RNA primers with a 3' G residue. Hantavirus RdRp has an N-terminal endonuclease domain and a C-terminal uncharacterized domain that harbors a binding site for the N protein. The purified endonuclease domain of RdRp nonspecifically degraded RNA in vitro It is puzzling how such nonspecific endonuclease activity generates primers of appropriate length and specificity during cap snatching. We fused the N-terminal endonuclease domain with the C-terminal uncharacterized domain of the RdRp. The resulting NC mutant, with the assistance of N protein, generated capped primers of appropriate length and specificity from a test mRNA in cells. Bacterially expressed and purified NC mutant and N protein required further incubation with the lysates of human umbilical vein endothelial cells (HUVECs) for the specific endonucleolytic cleavage of a test mRNA to generate capped primers of appropriate length and defined 3' terminus in vitro Our results suggest that an unknown host cell factor facilitates the interaction between N protein and NC mutant and brings the N protein-bound capped RNA fragments in close proximity to the endonuclease domain of the RdRp for specific cleavage at a precise length from the 5' cap. These studies provide critical insights into the cap-snatching mechanism of cytoplasmic viruses and have revealed potential new targets for their therapeutic intervention.IMPORTANCE Humans acquire hantavirus infection by the inhalation of aerosolized excreta of infected rodent hosts. Hantavirus infections cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), with mortality rates of 15% and 50%, respectively (1). Annually 150,000 to 200,000 cases of hantavirus infections are reported worldwide, for which there is no treatment at present. Cap snatching is an early event in the initiation of virus replication in infected hosts. Interruption in cap snatching will inhibit virus replication and will likely improve the prognosis of the hantavirus disease. Our studies provide mechanistic insight into the cap-snatching mechanism and demonstrate the requirement of a host cell factor for successful cap snatching. Identification of this host cell factor will reveal a novel therapeutic target for combating this viral illness.

Small RNAs in Rat Sperm Are a Predictive and Sensitive Biomarker of Exposure to the Testicular Toxicant Ethylene Glycol Monomethyl Ether.

Testicular histology and semen parameters are considered the gold standards when determining male reproductive toxicity. Ethylene glycol monomethyl ether (EGME) is a testicular toxicant with well-described effects on histopathology and sperm parameters. To compare the predictivity and sensitivity of molecular biomarkers of testicular toxicity to the traditional endpoints, small RNAs in the sperm were analyzed by next generation RNA-sequencing (RNA-seq). Adult rats were exposed to 0, 50, 60, or 75 mg/kg EGME by oral gavage for 5 consecutive days. Testis histology, epididymal sperm motility, and sperm small RNAs, including microRNAs (miRNAs), mRNA fragments, piwi-interacting RNAs (piRNAs), and tRNA fragments (tRFs), were analyzed 5 weeks after cessation of exposure. Testicular histology showed a significant dose-dependent increase in retained spermatid heads (RSH), while sperm motility declined with increasing dose. RNA-sequencing of sperm small RNAs was used to identify significant dose-dependent changes in percent mRNA fragments (of total reads), percent miRNAs (of total reads), average tRF length, average piRNA length, and piRNA and tRF length-distributions. Discriminant analysis showed relatively low predictivity of exposure based on RSH or motility compared to the average read length of all assigned RNAs. Benchmark dose (BMD) modeling resulted in a BMD of 62 mg/kg using RSH, whereas average read length of all assigned RNAs resulted in a BMD of 47 mg/kg. These results showed that sperm small RNAs are sensitive and predictive biomarkers of EGME-induced male reproductive toxicity.

Abstract B046: Immune repertoire sequencing enables complete B-cell and T-cell clonality determination and minimal residual disease assessment

Abstract The study of complex immunologic diseases and tumor microenvironment has progressed through recent developments that enable the sequencing of the immune repertoire. Using this approach, the interrogation of disease progression is facilitated through analysis of millions of V(D)J combinations from both B cell antibodies (Igs) and T-cell receptors (TCRs). One major challenge of immune repertoire sequencing is to capture the structural and sequence complexities of antibody and TCR genes. We have developed and optimized a method for accurate sequencing of full-length immune gene repertoires of B-cells and T-cells. RNA extracted from tissue and PBMCs were used to generate immune sequencing libraries. Using a unique molecule index (UMI) to discretely barcode each mRNA molecule, PCR copies of each mRNA fragment can be collapsed into a single consensus sequence. The B cell genes were enriched during library preparation by IGH, IGK and IGL primes, including isotype-specific primers (IgA, IgD, IgE, IgG and IgM). The T-cell genes were enriched by TCRα and TCRβ specific primers. To investigate the applicability of minimal residual disease assessment, we obtained Jurkat RNA, a homogenous population of leukemic Jurkat T-cells and spiked it into a PBMC RNA sample at varying proportions of Jurkat RNA (10%, 1%, 0.1%, 0.01% and 0.001%). The RNA mixtures were made into TCR libraries, sequenced on both Illumina MiSeq and Oxford Nanopore MinION, and analyzed to assess the TCR repertoire. Utilization of UMIs enabled absolute quantification of the B cell antibody/TCR clones and accurate ranking of their abundance. For B cell repertoire sequencing, the use of isotype-specific primers enabled measurement of the heavy chain isotype proportions within the samples. Full-length heavy chain antibody analysis enabled measurement of the mutation level of each antibody sequence, providing information on the overall maturity and mutational profile of the sample repertoire. For TCR repertoire sequencing, distinct and shared clonal sequences were quantitatively detected in PBMC samples. The method also accurately and sensitively detected the control TCR clone spiked into both Illumina and Oxford Nanopore libraries at levels appropriate for minimal residual disease assessment.This immune repertoire sequencing approach allows accurate clonal determination for both Igs and TCRs. This technique is applicable for a variety of applications including design of antibody chains for in vitro synthesis, investigation of T-cell infiltration of tumor microenvironments, and monitoring of minimal residual disease. Citation Format: Chen Song, Luo Sun, Pingfang Liu, Bradley Langhorst, Andrew Barry, Theodore B. Davis, Eileen T. Dimalanta. Immune repertoire sequencing enables complete B-cell and T-cell clonality determination and minimal residual disease assessment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B046.

High-Resolution Ribosome Profiling Defines Discrete Ribosome Elongation States and Translational Regulation during Cellular Stress

Ribosomes undergo substantial conformational changes during translation elongation to accommodate incoming aminoacyl-tRNAs and translocate along the mRNA template. We used multiple elongation inhibitors and chemical probing to define ribosome conformational states corresponding to differently sized ribosome-protected mRNA fragments (RPFs) generated by ribosome profiling. We show, using various genetic and environmental perturbations, that short 20-22 or classical 27-29 nucleotide RPFs correspond to ribosomes with open or occupied ribosomal A sites, respectively. These distinct states of translation elongation are readily discerned by ribosome profiling in all eukaryotes we tested, including fungi, worms, and mammals. This high-resolution ribosome profiling approach reveals mechanisms of translation-elongation arrest during distinct stress conditions. Hyperosmotic stress inhibits translocation through Rck2-dependent eEF2 phosphorylation, whereas oxidative stress traps ribosomes in a pre-translocation state, independent of Rck2-driven eEF2 phosphorylation. These results provide insights and approaches for defining the molecular events that impact translation elongation throughout biology.

Ribosome Profiling of Vaccinia Virus-Infected Cells.

Ribosome profiling is a method that determines genome-wide mRNA translation through measuring ribosome-protected mRNA fragments by deep sequencing. This method can be used to quantify gene expression at the translational level and precisely pinpoint ribosome loading onto mRNA with codon-level resolution. Genome-wide regulation of mRNA translation can also be determined if RNA-Sequencing (RNA-Seq) is carried out in parallel. Here, we describe a protocol for simultaneously performing ribosome profiling and RNA-Seq in cells infected with vaccinia virus.

Protocol for Ribosome Profiling in Bacteria.

Ribosome profiling provides information on the position of ribosomes on mRNA on a genomic scale. Although this information is often used to detect changes in gene expression under different conditions, it also has great potential for yielding insight into the mechanism and regulation of protein synthesis itself. First developed in yeast, ribosome profiling involves the isolation and sequencing of ribosome-protected mRNA fragments generated by nuclease treatment. Since the application of ribosome profiling in bacteria has been problematic, we report here a systematically optimized protocol for E. coli that we have used with success for other bacteria as well. Cells are harvested by flash-freezing cultures directly in liquid nitrogen. After lysis, translation is arrested by high magnesium concentration without the use of antibiotics. These improvements eliminate artifacts induced by harvesting cells by centrifugation or filtration and by use of chloramphenicol to arrest translation. These improvements are especially appropriate for studies where the exact position of the ribosome is critical, and not merely the number of ribosomes per message, such as studies aimed at monitoring differences in local elongation rates.

Polymerase-amplified release of ATP (POLARA) for detecting single nucleotide variants in RNA and DNA.

The identification of single nucleotide polymorphisms (SNP) is increasingly important for diagnosis and treatment of disease. Here we studied the potential use of ATP-releasing nucleotides (ARNs) for identifying SNPs in DNA and RNA targets. Synthesized as derivatives of the four canonical deoxynucleotides, ARNs can be used in the place of deoxynucleoside triphosphates to elongate a primer hybridized to a nucleic acid template, with the leaving group being ATP rather than pyrophosphate. The released ATP is then harnessed in conjunction with luciferase to generate chemiluminescence. Extension on a long target DNA or RNA generates many equivalents of ATP per target strand, providing isothermal amplification of signal. In principle, allele-specific primers could be used in conjunction with ARNs to generate differential luminescence signals with respect to distinct genetic polymorphisms. To test this, varied primer designs, modifications, enzymes and conditions were tested, resulting in an optimized strategy that discriminates between differing nucleic acid templates with single nucleotide resolution. This strategy was then applied to diagnostically relevant alleles resulting in discrimination between known polymorphisms. SNP detection was successfully performed on transcribed mRNA fragments from four different alleles derived from JAK2, BCR-ABL1, BRAF, and HBB. To investigate background interference, wild-type and mutant transcripts of these four alleles were tested and found to be easily distinguishable amid total cellular RNA isolated from human blood. Thus, ARNs have been employed with specialized allele-specific primers to detect diagnostically important SNPs in a novel method that is sensitive, rapid, and isothermal.

Optimization of ribosome profiling using low-input brain tissue from fragile X syndrome model mice.

Dysregulated protein synthesis is a major underlying cause of many neurodevelopmental diseases including fragile X syndrome. In order to capture subtle but biologically significant differences in translation in these disorders, a robust technique is required. One powerful tool to study translational control is ribosome profiling, which is based on deep sequencing of mRNA fragments protected from ribonuclease (RNase) digestion by ribosomes. However, this approach has been mainly applied to rapidly dividing cells where translation is active and large amounts of starting material are readily available. The application of ribosome profiling to low-input brain tissue where translation is modest and gene expression changes between genotypes are expected to be small has not been carefully evaluated. Using hippocampal tissue from wide type and fragile X mental retardation 1 (Fmr1) knockout mice, we show that variable RNase digestion can lead to significant sample batch effects. We also establish GC content and ribosome footprint length as quality control metrics for RNase digestion. We performed RNase titration experiments for low-input samples to identify optimal conditions for this critical step that is often improperly conducted. Our data reveal that optimal RNase digestion is essential to ensure high quality and reproducibility of ribosome profiling for low-input brain tissue.

Detection of MicroRNA-Mediated Target mRNA Cleavage and 3'-Uridylation in Human Cells by a SLA-RT-PCR Analysis.

MicroRNA (miRNA) plays an important role in posttranscriptional regulation of gene expression by dominantly binding to the 3'-UTR regions of target mRNAs in the miRNA-induced silencing complex (miRISC), triggering off their sequential cleavage and 3'-uridylation, facilitating their degradation, repressing target gene expression, and leading to a reduced protein output. The miRNA-mediated target mRNA cleavage activity generates cleaved mRNA fragments with varied termini, which creates major technical challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs and the resulting mRNA fragments in transition. Here we described a sensitive stem-loop array reverse transcription polymerase chain reaction (SLA-RT-PCR) approach to detect and verify the miRNA-mediated target mRNA cleavage sites by determining precise sequences at the 3'- termini of cleaved mRNA fragments and their 3'-uridylation in human cells under physiological conditions. The SLA-RT-PCR methods have been demonstrated as a sensitive, cost-efficient, and high-throughput tool to systematically detect miRNA-targeted mRNA cleavage sites and fragments with 3'-uridylation in human cells.

Recovery and analysis of transcriptome subsets from pooled single-cell RNA-seq libraries.

Single-cell RNA sequencing (scRNA-seq) methods generate sparse gene expression profiles for thousands of single cells in a single experiment. The information in these profiles is sufficient to classify cell types by distinct expression patterns but the high complexity of scRNA-seq libraries often prevents full characterization of transcriptomes from individual cells. To extract more focused gene expression information from scRNA-seq libraries, we developed a strategy to physically recover the DNA molecules comprising transcriptome subsets, enabling deeper interrogation of the isolated molecules by another round of DNA sequencing. We applied the method in cell-centric and gene-centric modes to isolate cDNA fragments from scRNA-seq libraries. First, we resampled the transcriptomes of rare, single megakaryocytes from a complex mixture of lymphocytes and analyzed them in a second round of DNA sequencing, yielding up to 20-fold greater sequencing depth per cell and increasing the number of genes detected per cell from a median of 1313 to 2002. We similarly isolated mRNAs from targeted T cells to improve the reconstruction of their VDJ-rearranged immune receptor mRNAs. Second, we isolated CD3D mRNA fragments expressed across cells in a scRNA-seq library prepared from a clonal T cell line, increasing the number of cells with detected CD3D expression from 59.7% to 100%. Transcriptome resampling is a general approach to recover targeted gene expression information from single-cell RNA sequencing libraries that enhances the utility of these costly experiments, and may be applicable to the targeted recovery of molecules from other single-cell assays.