Publications by authors named "Laura M Agosto"

4 Publications

  • Page 1 of 1

Deep profiling and custom databases improve detection of proteoforms generated by alternative splicing.

Genome Res 2019 12 14;29(12):2046-2055. Epub 2019 Nov 14.

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Alternative pre-mRNA splicing has long been proposed to contribute greatly to proteome complexity. However, the extent to which mature mRNA isoforms are successfully translated into protein remains controversial. Here, we used high-throughput RNA sequencing and mass spectrometry (MS)-based proteomics to better evaluate the translation of alternatively spliced mRNAs. To increase proteome coverage and improve protein quantitation, we optimized cell fractionation and sample processing steps at both the protein and peptide level. Furthermore, we generated a custom peptide database trained on analysis of RNA-seq data with MAJIQ, an algorithm optimized to detect and quantify differential and unannotated splice junction usage. We matched tandem mass spectra acquired by data-dependent acquisition (DDA) against our custom RNA-seq based database, as well as SWISS-PROT and RefSeq databases to improve identification of splicing-derived proteoforms by 28% compared with use of the SWISS-PROT database alone. Altogether, we identified peptide evidence for 554 alternate proteoforms corresponding to 274 genes. Our increased depth and detection of proteins also allowed us to track changes in the transcriptome and proteome induced by T-cell stimulation, as well as fluctuations in protein subcellular localization. In sum, our data here confirm that use of generic databases in proteomic studies underestimates the number of spliced mRNA isoforms that are translated into protein and provides a workflow that improves isoform detection in large-scale proteomic experiments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.248435.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886501PMC
December 2019

RNA Binding Protein CELF2 Regulates Signal-Induced Alternative Polyadenylation by Competing with Enhancers of the Polyadenylation Machinery.

Cell Rep 2019 09;28(11):2795-2806.e3

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Graduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

The 3' UTR (UTR) of human mRNAs plays a critical role in controlling protein expression and function. Importantly, 3' UTRs of human messages are not invariant for each gene but rather are shaped by alternative polyadenylation (APA) in a cell state-dependent manner, including in response to T cell activation. However, the proteins and mechanisms driving APA regulation remain poorly understood. Here we show that the RNA-binding protein CELF2 controls APA of its own message in a signal-dependent manner by competing with core enhancers of the polyadenylation machinery for binding to RNA. We further show that CELF2 binding overlaps with APA enhancers transcriptome-wide, and almost half of 3' UTRs that undergo T cell signaling-induced APA are regulated in a CELF2-dependent manner. These studies thus reveal CELF2 to be a critical regulator of 3' UTR identity in T cells and demonstrate an additional mechanism for CELF2 in regulating polyadenylation site choice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.08.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6752737PMC
September 2019

One minute analysis of 200 histone posttranslational modifications by direct injection mass spectrometry.

Genome Res 2019 06 23;29(6):978-987. Epub 2019 May 23.

Epigenetics Institute, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

DNA and histone proteins define the structure and composition of chromatin. Histone posttranslational modifications (PTMs) are covalent chemical groups capable of modeling chromatin accessibility, mostly due to their ability in recruiting enzymes responsible for DNA readout and remodeling. Mass spectrometry (MS)-based proteomics is the methodology of choice for large-scale identification and quantification of protein PTMs, including histones. High sensitivity proteomics requires online MS coupling with relatively low throughput and poorly robust nano-liquid chromatography (nanoLC) and, for histone proteins, a 2-d sample preparation that includes histone purification, derivatization, and digestion. We present a new protocol that achieves quantitative data on about 200 histone PTMs from tissue or cell lines in 7 h from start to finish. This protocol includes 4 h of histone extraction, 3 h of derivatization and digestion, and only 1 min of MS analysis via direct injection (DI-MS). We demonstrate that this sample preparation can be parallelized for 384 samples by using multichannel pipettes and 96-well plates. We also engineered the sequence of a synthetic "histone-like" peptide to spike into the sample, of which derivatization and digestion benchmarks the quality of the sample preparation. We ensure that DI-MS does not introduce biases in histone peptide ionization as compared to nanoLC-MS/MS by producing and analyzing a library of synthetically modified histone peptides mixed in equal molarity. Finally, we introduce EpiProfileLite for comprehensive analysis of this new data type. Altogether, our workflow is suitable for high-throughput screening of >1000 samples per day using a single mass spectrometer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.247353.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581051PMC
June 2019

Alternative pre-mRNA splicing switch controls hESC pluripotency and differentiation.

Genes Dev 2018 09;32(17-18):1103-1104

Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA.

Alternative splicing (AS) of pre-mRNAs is a ubiquitous process in mammals that is tightly regulated in a cell type- and cell state-dependent manner. However, the details of how splicing is regulated to impact specific cell fate decisions remains incompletely understood. A study by Yamazaki and colleagues (pp. 1161-1174) in this issue of provides exciting new insight into the role and regulation of splicing in the maintenance of pluripotency of human embryonic stem cells (hESCs). In brief, they show that AS of several genes is robustly regulated upon differentiation of hESCs. One of these genes, T-cell factor 3 (), is regulated at least in part through the activity of heterogeneous nuclear ribonucleoproteins H1 and F (hnRNP H/F) to control the mutually exclusive expression of the encoded E12 and E47 transcription regulators. The investigators demonstrate that reduced expression of hnRNP H/F favors expression of E47, which in turn decreases E-cadherin expression to promote hESC differentiation. In contrast, high levels of hnRNP H/F induce expression of E12 to maintain pluripotency. Thus, this work provides at least one new link between AS and control of human stem cell fate and suggests a broader role of splicing in pluripotency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.318451.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120719PMC
September 2018