DOI: 10.6078/d1h019
title: Transcriptome Analysis Reveals Extensive Alternative Splicing-Coupled Nonsense-Mediated mRNA Decay in a Human Cell Line

description: To further explore the regulatory potential of nonsense-mediated mRNA
 decay (NMD) in human cells, we globally surveyed the transcripts targeted
 by this pathway via RNA-Seq analysis of HeLa cells in which NMD had been
 inhibited. We first identified those transcripts with both a premature
 termination codon more than 50 nucleotides upstream of an exon-exon
 junction (50nt rule) and a significant increase in abundance upon NMD
 inhibition. Remarkably, at least 2,793 transcripts derived from 2,116
 genes are physiological NMD targets (9.2% of expressed transcripts and
 &gt;20% of alternatively spliced genes). Our analysis identifies
 previously inferred unproductive isoforms and numerous previously
 uncharacterized ones. NMD-targeted transcripts were derived from genes
 involved in many functional categories, and are particularly enriched for
 RNA splicing genes and ultraconserved elements. By investigating the
 features of all transcripts impacted by NMD, we find that the 50nt rule is
 a strong predictor of NMD degradation while 3’ UTR length generally has
 only a small effect in human cells. Additionally, thousands more
 transcripts without a premature termination codon upstream of an exon-exon
 junction in the main coding sequence contain a uORF and display
 significantly increased abundance upon NMD inhibition indicating
 potentially widespread regulation through decay coupled with uORF
 translation. Our results support the hypothesis that alternative splicing
 coupled with NMD is a prevalent post-transcriptional mechanism in human
 cells with broad potential for biological regulation.
description: HeLa cells were inoculated on plates in Dulbecco’s MEM medium with 0.1 mM
 non-essential amino acids and 10% fetal bovine serum, incubated at 37°C
 and 5% CO2. Plates at 80% cell confluency were transfected with plasmids
 pSUPERpuro-hUpf1/II and pSUPERpuro-Scramble, whose functions were to
 knockdown UPF1 and act as a mock control, respectively. Transfections were
 performed using Lipofectamine™ LTX and PLUS™ Reagents (Invitrogen)
 according to the manufacturer’s protocol, and the following culture
 according to the published method (Paillusson et al., 2005). The whole
 cell lysates were prepared in 1% sodium dodecyl sulfate and incubated at
 100 °C for 5 min, then centrifuged at 12,000 rpm for 10 min. Total RNA was
 extracted using the QIAGEN RNeasy® Mini kit according to the
 manufacturer’s manual. Directional and paired-end RNA-seq libraries were
 constructed according to the protocol published on the Illumina website
 (www.illumina.com), with a few changes: The adapters were prepared
 according to the reported methods (Vigneault et al., 2008). The PCR
 process to prepare the library was divided in two steps. In the first
 step, 3 cycles of PCR were performed (according to the protocol) to
 prepare the library template, and then the library was run on a 2% agarose
 gel, fragments of desired size were cut out and isolated by QIAquick® Gel
 Extraction Kit. A second round of PCR (12 cycles) was performed to enrich
 the library and then it was purified twice with Agencourt AMPure XP kit as
 suggested by the Illumina protocol. The libraries were then assayed by
 Agilent 2100 BioAnalyzer. These RNA-Seq libraries were prepared from cells
 with inhibited NMD and control cells for two biological replicates. One
 biological replicate was sequenced on an Illumina GAIIx machine and the
 other on HiSeq 2000. 
 
 Paired end reads for each library were aligned to the NCBI human RefSeq
 transcriptome (Pruitt et al., 2009) with Bowtie (Langmead et al., 2009) to
 determine the average insert size and standard deviation, required as a
 parameter by TopHat (Trapnell et al., 2009). The reads of each library
 were then aligned to the human genome (hg19 assembly, Feb. 2009;
 downloaded from UCSC genome browser (Fujita et al., 2011)) using TopHat
 v1.2.0 with default parameters plus the following: --coverage search,
 --allow indels, --microexon search, and --butterfly search. Cufflinks
 1.0.1 (Roberts et al., 2011; Trapnell et al., 2010) was used to assemble
 each set of aligned reads into transcripts with the UCSC known transcript
 set (Fujita et al., 2011) as the reference guide, along with the following
 parameters: --frag-bias-correct, and --multi-read-correct. Cuffcompare (a
 sub-tool of Cufflinks) was used to merge the resulting sets of assembled
 transcripts. Each junction was assigned a Shannon entropy score based on
 offset of spliced reads across all four libraries. Transcripts with a
 junction that had an entropy score &lt;1 and was not present in the
 reference annotation were filtered out. Cuffdiff (a sub-tool of Cufflinks)
 was used to quantify and compare transcript abundance (measured by FPKM,
 Fragments Per Kilobase per Million reads) between the UPF1 knockdown and
 control samples. For each sample, the reads from two biological replicates
 were provided. The following parameters were used: --frag-bias-correct and
 --multi-read-correct. Only transcripts with FPKM&gt;1 in either the
 control or UPF1 knockdown sample were used for further analysis. A
 transcript was called significantly more abundant in the UPF1 knockdown
 sample if Cuffdiff called it significantly changing and the fold change
 was greater than 1.5x. Significantly decreased transcript abundances were
 determined in the same way. For each transcript, the coding sequence (CDS)
 was determined as described in the Supplementary Methods. A coding
 sequence was defined to terminate in a premature stop codon (PTC50nt) if
 it stops at least 50 nucleotides upstream of the last exon-exon junction
 (50nt rule in mammals (Nagy and Maquat, 1998)). NMD targets were defined
 as those transcripts with both a PTC50nt and significantly increased
 expression abundance in NMD inhibited (UPF1 knockdown) cells. The
 transcripts must also increase in each biological replicate when analyzed
 independently and come from a gene with a non-PTC50nt-containing isoform
 with FPKM&gt;0. To obtain a more reliable list of NMD-targeted
 transcripts, only those transcripts that adhered to either of the
 following criteria were kept: 1) No non-PTC50nt-containing isoform from
 the gene was more than 1.2-fold higher in the NMD inhibited sample, or 2)
 the PTC50nt-containing isoform increased at least 2x more than the sum of
 all non-PTC50nt-containing isoform FPKMs from the gene in NMD inhibited
 cells.