Project description:Eukaryotic genes generate multiple mRNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5′ and 3′ untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5′ untranslated regions exert robust translational control between cell lines, while 3′ untranslated regions can confer cell-type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels.
Project description:To understand the effect of FUS protein knock-down on poly(A+) RNAs profiles, we carried out control and FUS depletion (using synthetic siRNAs) experiments followed by poly(A+) RNA 3'-seq anlyais using lexogen kit (016.024)in HeLa cells
Project description:Regulation of presenilin genes. Presenilins are intramembrane aspartic proteases. These proteases are critical proteins in pathogenesis of Alzheimer's disease. The function of recently identified presenilin-homologous proteases (IMPAS or SPP)s is unknown. Our preliminary data in C.elegans model suggested the role of these proteins in early-development and , perhaps, in pathway related- to cholesterol -regulated signalling (Grigorenko et al, 2004, PNAS). The overall goal is to determine pattern of gene expression alterations in cells with knock-out or knock-down presenilin related proteins (IMPAS). The cultured cells, C.elegans and mouse models are planned to be used in this study. Initially, we will determine gene expression patterns in human cells embryonic kidney cells (HEK 293 cell line) overexpressing different isoforms of hIMP1 protein. We hypothesize that novel family of presenilin-related proteases is important for cholesterol-regulated intracellular signalling and early development (including CNS/neuronal development and function). We will examine in HEK 293 cells effect of over expression of 1) hIMP1 wt protein, 2) over expression of dominant negative isoform of hIMP1 protein and compare their gene expression patterns to 3) HEK 293 cells treated with mock vector. HEK 293 cells were seeded on 10 cm plates and transfected next day using Lipofectamine Plus reagent. 24 hours after cells were washed twice with ice cold PBS buffer and lysed with TRizol reagent directly on plates. Total RNA was extracted, purified according the manufacturer protocol and stored at -800C. We will provide 2 RNA samples for each hIMP1 isoform used in two different transfection experiment to reduce any spurious expression differences resulting from culture condition or transfection efficiency variations.
Project description:Eukaryotic genes generate multiple mRNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5â?² and 3â?² untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5â?² untranslated regions exert robust translational control between cell lines, while 3â?² untranslated regions can confer cell-type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels. Total cytoplasmic and eight polysomal fractions of RNA were purified from HEK 293T cells in biological duplicate. Ribosomal RNA was depleted using Ribo-Zero (Human/Mouse/Rat; Epicenter) and libraries were prepared using the TruSeq RNA v2 kit (RS-122-2001; Illumina) skipping the polyA selection step. Reads are paired-end 75bp and sequencing adapters are GATCGGAAGAGCACACGTCTGAACTCCAGTCAC (read1) and AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT (read2).
Project description:Alternative splicing is a key mechanism for expanding transcriptomic and proteomic complexity, yet its role in innate immune activation remains incompletely understood. Here, we applied Oxford Nanopore native RNA sequencing to generate an isoform-level transcriptome of primary human monocytes before and after activation with lipopolysaccharide (LPS). We identify over 24,000 expressed isoforms, including thousands of previously unannotated variants. Activation with LPS induced widespread isoform-specific expression changes, leading to extensive isoform switching events, which were validated using matched short-read RNA-Seq. These activation-induced shifts enhanced transcript immune-regulatory functions: activated monocytes preferentially express longer, coding-competent isoforms with complete open reading frames, fewer retained introns, and increased domain complexity. By integrating matched Ribo-seq and proteomic data, we demonstrate that these isoform modulations are associated with enhanced translation of immune effector proteins. Together, our findings position alternative splicing as a dynamic and functional regulator of monocyte activation, emphasizing the need for isoform-level resolution to fully understand immune cell function and inflammation.
Project description:To understand the global effects of B2M knock-down on host gene expression, the RNA-seq study was carried out using HSV-1 infected HeLa cells transfected with B2M siRNAs (siB2M) or negative control siRNAs (siCTRL)