Untranslated, maybe, unappreciated, no more.

From the Hsieh Lab, Human Biology Division

Genomic DNA encompassing protein coding sequences also contains regulatory sequences such as promoters, introns, and enhancers as well as regions that seemingly serve little or no purpose.  When genomic DNA is transcribed, many of the non-coding sequences are omitted or spliced out.  The resulting messenger RNA (mRNA) contains the intact protein coding sequence flanked by 5’ and 3’ untranslated regions (UTRs).  The UTRs, ranging in size from 20 or so bases to several kilobases, have been studied extensively and regulatory functions impacting mRNA abundance and stability, mRNA structure and translation efficiency have been catalogued.  The impacts of polymorphisms or mutations on abundance, stability and translation efficiency of mRNAs encoding cancer-related proteins including c-Myc, BRCA1 and RB1 have also been noted.  What has been lacking is a comprehensive way to examine the functional impacts of polymorphisms or mutations in full-length 5’-UTR on a transcriptome-wide scale.  Until now. 

Post-doctoral fellow Yiting Lim and colleagues in Andrew Hsieh’s laboratory in the Human Biology Division along with Fred Hutch/UW Cancer Consortium collaborators Lawrence True, Eva Corey, Peter Nelson and Gavin Ha developed a functional genomic methodology to meet this challenge taking advantage of the extensive prostate cancer patient tissue repository and computational resources of the PNW Prostate Cancer Specialized Program of Research Excellence (SPORE). The research also would not have been possible without the Cancer Consortium’s genomic core and access to both long-read and short-read sequencing technologies. The method, called Pooled full-length UTR Multiplex Assay on Gene Expression (PLUMAGE) is an massively parallel reporter assay that couples long-read and short-read sequencing technologies to measure the impact of somatic mutations with 5’ UTRs at both the transcript and translation levels simultaneously. Writing in the journal Nature Communications, the authors describe the methodology and apply it to the analysis of patient samples from 229 localized and metastatic prostate cancer samples.

Non-coding regions of genes influence prostate cancer pathways.
Non-coding regions of genes influence prostate cancer pathways. Image provided by Dr. Yiting Lim, Hsieh group.

The researchers started with an analysis of five well-characterized, patient-derived primary cancer cell lines representing the breadth of genetic and phenotypic diversity in prostate cancer.  They performed high throughput, exome sequencing to identify 5’-UTR alterations.  To determine if these mutations were associated with changes in gene expression, they conducted transcriptome profiling and sequencing of ribosome-bound mRNAs from the same tissues.  Importantly, 12-40% of the 5’-UTR alterations per PDX line were associated with changes in transcript level and a similar percentage of alterations impacted mRNA translation.  To confirm that the observed changes were due to UTR alterations, the authors performed a mini-pooled screen. Libraries containing 5’ UTRs of three genes, ADAM32, COMT and ZCCHC7, each with a single nucleotide change representing increased, decreased or unchanged mRNA translation, respectively, were linked with bar-codes and assayed for changes in translation. This pooled assay which formed the basis of PLUMAGE confirmed the tissue-based findings and also orthogonal luciferase assays.

Having validated the ability of PLUMAGE to catalogue 5’-UTR alterations and determine their impact mRNA levels and their translation, the researchers turned their attention to a whole genome analysis of 229 patients.  To this end, sequencing results for 149 localized and 80 metastatic prostate cancer samples were interrogated using WGS or 5’UTR-seq.  2200 single nucleotide variants in 1878 genes were identified.  As expected, localized prostate cancer samples contained fewer variants than samples from metastatic cancers. To determine the functionality of these mutations, the author performed PLUMAGE on all recurrently mutated genes identified in these patients and discovered that 35% of mutations impacted either transcript or translation levels.

Seeking to identify mechanisms by which 5’-UTR changes exerted their effects, Lim and colleagues cross-referenced their prostate cancer alterations with RNA and DNA regulatory motif databases.  Their analysis showed that alterations tended to cluster near cis-acting RNA regulatory sequences as well as at or near DNA binding cites.  Most striking of the later was the creation of an E-box Myc binding site upstream of the fibroblast growth factor 7 (FGF7) gene that led to a significant upregulation of the corresponding mRNA.  Binding of MYC/MAX dimers to the mutant but not wild type DNA corresponding to the variant 5’-UTR.  A critical advantage of the PLUMAGE methodology is its ability to identify variants that exert biologically significant effects regardless of the specific mechanism of action. 

The authors also correlated their PLUMAGE hits with patient outcomes data. They found that patient tissues with functional mutations to MAP kinase pathway genes exhibited an increase in MAP kinase gene expression signatures. Furthermore, they were more likely to respond to taxane based chemotherapy. The MAP kinase signaling pathway has been associated with metastasis in murine models of prostate cancer. Analyzing all patients with MAP kinase pathway 5’ UTR mutations the authors found that patients with these mutations were twice and likely to present at diagnosis with metastatic prostate cancer. They conclude that 5’ UTR mutations may be associate with specific clinical outcomes.

According to Hsieh “Our work is the first to demonstrate that patient-based 5’ UTR can functionally impact oncogenic or tumor suppressive programs in cancer and have clinical implications for patients.”  Additionally, the demonstration that PLUMAGE can be applied to a high throughput analysis of full-length UTRs opens the door to evaluation of UTR changes across many if not all cancer types.  

This research was supported by grants from the National Institutes of Health, the Prostate Cancer Research Foundation, Burroughs Wellcome Fund, Robert J. Kleberg Jr. and Helen C. Kleberg Foundation, an AACR-Bristol Myers Squibb Oncology Fellowship and the Department of Defense CDMRP Prostate Cancer Training Award. 

Fred Hutch/UW Cancer Consortium member Andrew Hsieh led this study with contributions from members Eva Corey, Lawrence True, Peter Nelson and Gavin Ha.

Lim Y, Arora S, Schuster SL, Corey L, Fitzgibbon M, Wladyka CL, Wu X, Coleman IM, Delrow JJ, Corey E, True LD, Nelson PS, Ha G, Hsieh AC. Multiplexed functional genomic analysis of 5' untranslated region mutations across the spectrum of prostate cancer. Nat Commun. 2021 Jul 9;12(1):4217. doi: 10.1038/s41467-021-24445-6. PMID: 34244513; PMCID: PMC8270899.