Current Status and Future Opportunities in Lung Precision Medicine Research with a Focus on Biomarkers. An American Thoracic Society/National Heart, Lung, and Blood Institute Research Statement.

BACKGROUND: Thousands of biomarker tests are either available or under development for lung diseases. In many cases, adoption of these tests into clinical practice is outpacing the generation and evaluation of sufficient data to determine clinical utility and ability to improve health outcomes. There is a need for a systematically organized report that provides guidance on how to understand and evaluate use of biomarker tests for lung diseases. METHODS: We assembled a diverse group of clinicians and researchers from the American Thoracic Society and leaders from the National Heart, Lung, and Blood Institute with expertise in various aspects of precision medicine to review the current status of biomarker tests in lung diseases. Experts summarized existing biomarker tests that are available for lung cancer, pulmonary arterial hypertension, idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, sepsis, acute respiratory distress syndrome, cystic fibrosis, and other rare lung diseases. The group identified knowledge gaps that future research studies can address to efficiently translate biomarker tests into clinical practice, assess their cost-effectiveness, and ensure they apply to diverse, real-life populations. RESULTS: We found that the status of biomarker tests in lung diseases is highly variable depending on the disease. Nevertheless, biomarker tests in lung diseases show great promise in improving clinical care. To efficiently translate biomarkers into tests used widely in clinical practice, researchers need to address specific clinical unmet needs, secure support for biomarker discovery efforts, conduct analytical and clinical validation studies, ensure tests have clinical utility, and facilitate appropriate adoption into routine clinical practice. CONCLUSIONS: Although progress has been made toward implementation of precision medicine for lung diseases in clinical practice in certain settings, additional studies focused on addressing specific unmet clinical needs are required to evaluate the clinical utility of biomarkers; ensure their generalizability to diverse, real-life populations; and determine their cost-effectiveness.

Phospho-site mutants of the RNA Polymerase II C-terminal domain alter subtelomeric gene expression and chromatin modification state in fission yeast.

Eukaryotic gene expression requires that RNA Polymerase II (RNAP II) gain access to DNA in the context of chromatin. The C-terminal domain (CTD) of RNAP II recruits chromatin modifying enzymes to promoters, allowing for transcription initiation or repression. Specific CTD phosphorylation marks facilitate recruitment of chromatin modifiers, transcriptional regulators, and RNA processing factors during the transcription cycle. However, the readable code for recruiting such factors is still not fully defined and how CTD modifications affect related families of genes or regional gene expression is not well understood. Here, we examine the effects of manipulating the Y1S2P3T4S5P6S7 heptapeptide repeat of the CTD of RNAP II in Schizosaccharomyces pombe by substituting non-phosphorylatable alanines for Ser2 and/or Ser7 and the phosphomimetic glutamic acid for Ser7. Global gene expression analyses were conducted using splicing-sensitive microarrays and validated via RT-qPCR. The CTD mutations did not affect pre-mRNA splicing or snRNA levels. Rather, the data revealed upregulation of subtelomeric genes and alteration of the repressive histone H3 lysine 9 methylation (H3K9me) landscape. The data further indicate that H3K9me and expression status are not fully correlated, suggestive of CTD-dependent subtelomeric repression mechansims that act independently of H3K9me levels.

Novel features of ARS selection in budding yeast Lachancea kluyveri.

BACKGROUND: The characterization of DNA replication origins in yeast has shed much light on the mechanisms of initiation of DNA replication. However, very little is known about the evolution of origins or the evolution of mechanisms through which origins are recognized by the initiation machinery. This lack of understanding is largely due to the vast evolutionary distances between model organisms in which origins have been examined. RESULTS: In this study we have isolated and characterized autonomously replicating sequences (ARSs) in Lachancea kluyveri - a pre-whole genome duplication (WGD) budding yeast. Through a combination of experimental work and rigorous computational analysis, we show that L. kluyveri ARSs require a sequence that is similar but much longer than the ARS Consensus Sequence well defined in Saccharomyces cerevisiae. Moreover, compared with S. cerevisiae and K. lactis, the replication licensing machinery in L. kluyveri seems more tolerant to variations in the ARS sequence composition. It is able to initiate replication from almost all S. cerevisiae ARSs tested and most Kluyveromyces lactis ARSs. In contrast, only about half of the L. kluyveri ARSs function in S. cerevisiae and less than 10% function in K. lactis. CONCLUSIONS: Our findings demonstrate a replication initiation system with novel features and underscore the functional diversity within the budding yeasts. Furthermore, we have developed new approaches for analyzing biologically functional DNA sequences with ill-defined motifs.

Genome-wide approaches to monitor pre-mRNA splicing.

Pre-mRNA processing is an essential control-point in the gene expression pathway of eukaryotic organisms. The budding yeast Saccharomyces cerevisiae offers a powerful opportunity to examine the regulation of this pathway. In this chapter, we will describe methods that have been developed in our lab and others to examine pre-mRNA splicing from a genome-wide perspective in yeast. Our goal is to provide all of the necessary information--from microarray design to experimental setup to data analysis--to facilitate the widespread use of this technology.

Cwc23, an essential J protein critical for pre-mRNA splicing with a dispensable J domain.

J proteins are structurally diverse, obligatory cochaperones of Hsp70s, each with a highly conserved J domain that plays a critical role in the stimulation of Hsp70's ATPase activity. The essential protein, Cwc23, is one of 13 J proteins found in the cytosol and/or nucleus of Saccharomyces cerevisiae. We report that a partial loss-of-function CWC23 mutant has severe, global defects in pre-mRNA splicing. This mutation leads to accumulation of the excised, lariat form of the intron, as well as unspliced pre-mRNA, suggesting a role for Cwc23 in spliceosome disassembly. Such a role is further supported by the observation that this mutation results in reduced interaction between Cwc23 and Ntr1 (SPP382), a known component of the disassembly pathway. However, Cwc23 is a very atypical J protein. Its J domain, although functional, is dispensable for both cell viability and pre-mRNA splicing. Nevertheless, strong genetic interactions were uncovered between point mutations encoding alterations in Cwc23's J domain and either Ntr1 or Prp43, a DExD/H-box helicase essential for spliceosome disassembly. These genetic interactions suggest that Hsp70-based chaperone machinery does play a role in the disassembly process. Cwc23 provides a unique example of a J protein; its partnership with Hsp70 plays an auxiliary, rather than a central, role in its essential cellular function.

Unproductive splicing of SR genes associated with highly conserved and ultraconserved DNA elements.

The human and mouse genomes share a number of long, perfectly conserved nucleotide sequences, termed ultraconserved elements. Whereas these regions can act as transcriptional enhancers when upstream of genes, those within genes are less well understood. In particular, the function of ultraconserved elements that overlap alternatively spliced exons of genes encoding RNA-binding proteins is unknown. Here we report that in every member of the human SR family of splicing regulators, highly or ultraconserved elements are alternatively spliced, either as alternative 'poison cassette exons' containing early in-frame stop codons, or as alternative introns in the 3' untranslated region. These alternative splicing events target the resulting messenger RNAs for degradation by means of an RNA surveillance pathway called nonsense-mediated mRNA decay. Mouse orthologues of the human SR proteins exhibit the same unproductive splicing patterns. Three SR proteins have been previously shown to direct splicing of their own transcripts, and one of these is known to autoregulate its expression by coupling alternative splicing with decay; our results suggest that unproductive splicing is important for regulation of the entire SR family. We find that unproductive splicing associated with conserved regions has arisen independently in different SR genes, suggesting that splicing factors may readily acquire this form of regulation.

Identification of Lhp1p-associated RNAs by microarray analysis in Saccharomyces cerevisiae reveals association with coding and noncoding RNAs.

La is a conserved eukaryotic RNA-binding protein best known for its role in the biogenesis of noncoding RNAs transcribed by RNA polymerase III. To broaden our understanding of the function of the La homologous protein (Lhp1) in Saccharomyces cerevisiae, we have taken a genomics approach. Lhp1 ribonucleoprotein complexes were immunoprecipitated and bound RNAs were examined by hybridization to whole-genome microarrays that include >6,000 ORFs, documented noncoding RNAs, and the intervening intergenic regions. Demonstrating the validity of this approach, associations with previously known Lhp1p-associated RNAs were detected and associations with additional noncoding RNAs, including multiple tRNAs and small nucleolar RNAs, were revealed. Indicating that this approach provides a robust method for discovering RNAs, the data also identify associations between Lhp1p and several intergenic regions, three of which encode the recently annotated putative snoRNAs: RUF1, RUF2, and RUF3. Unexpectedly, we find that Lhp1p is also associated with a subset of coding mRNAs. These mRNAs include many ribosomal protein transcripts as well as the mRNA encoding Hac1p, a transcription factor required during the unfolded protein stress response. In cells lacking LHP1, Hac1p levels are decreased 2- to 3-fold, whereas no changes are detected in the levels of spliced or unspliced HAC1 mRNA or in the stability of Hac1p. Finally, although LHP1 is dispensable for growth under standard conditions, we find that it is required when the unfolded protein response is induced at elevated temperatures. These results suggest that Lhp1p may play a novel role in the translation of one or more cellular mRNAs.