We sought to comprehensively elucidate the transcriptomic underpinnings of microgravity-induced muscle phenotypes in mice by evaluating both differential gene expression (DGE) and changes in alternative splicing (AS) due to extended spaceflight. Total RNA was isolated from the gastrocnemius and quadriceps, postural and phasic muscles of the hind limb, respectively, of 32-week-old female BALB/c mice exposed to microgravity or ground control conditions for nine weeks. RNA sequencing revealed that DGE and AS varied across postural and phasic muscle types with preferential employment of DGE in the gastrocnemius and AS in the quadriceps. Gene ontology analysis indicated that DGE and AS regulate distinct molecular processes. Various non-differentially expressed transcripts encoding musculoskeletal proteins (Tnnt3, Tnnt1, Neb, Ryr1, and Ttn) and muscle-specific RNA binding splicing regulators (Mbnl1 and Rbfox1) were found to have significant changes in AS that altered critical functional domains of their protein products. In striking contrast, microgravity-induced differentially expressed genes were associated with translation/ribosomal function and lipid metabolism. Our work serves as the first comprehensive investigation of coordinate changes in DGE and AS in large limb muscles across spaceflight. We propose that substantial remodeling of pre-mRNA by AS is a major component of transcriptomic adaptation of skeletal muscle to microgravity. The alternatively spliced genes identified here could be targeted by small molecule splicing regulator therapies to address microgravity-induced changes in muscle during spaceflight.

Low RNA yield and quality limit use of formalin-fixed paraffin-embedded (FFPE) tissue samples for genomic analyses. In this study, we evaluated methods to demodify RNA highly fragmented and crosslinked by formalin fixation. Primary endpoints were RNA recovery, RNA-sequencing quality metrics, and target gene responses to a reference chemical (phenobarbital, PB). Frozen mouse liver samples from control and PB groups (n=6/group) were divided and preserved for 3 months as follows: frozen (FR); 70% ethanol (OH); 10% buffered formalin for 18 hours followed by ethanol (18F); and 10% buffered formalin (3F). Samples from OH, 18F, and 3F groups were processed to FFPE blocks and sectioned for RNA isolation. The latter group received no additional treatment (3F) or the following demodification protocols: short heated incubation with TAE buffer; overnight heated incubation with an organocatalyst using two different isolation kits; or overnight heated incubation without organocatalyst. TruSeq Stranded Total RNA libraries with Ribo-Zero were built and sequenced using the Illumina HiSeq platform. Extended incubation with or without organocatalyst increased RNA yield >3-fold and enhanced quality compared to 3F, as indicated by higher RNA integrity number (>1.5-fold) and fragment analysis values (>3.0-fold). Post-sequencing metrics showed reduced bias in gene coverage and deletion rates for all extended incubation groups. Following PB-induced differential gene expression analysis, all demodification groups showed increased overlap with FR in genes (73-83%) and pathways (91-94%) compared to 3F overlap with FR (60% and 63%, respectively). These results demonstrate simple changes in RNA isolation methods that can enhance genomic analyses of FFPE samples. This dataset is associated with the following publication: Wehmas, L., C. Wood, R. Gagne, A. Williams, C. Yauk, M. Gosink, D. Dalmas, R. Hao, R. O'Lone, and S. Hester. Demodifying RNA for Transcriptomic Analyses of Archival Formalin-Fixed Paraffin-Embedded Samples. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 162(2): 535-547, (2018).