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Despite their fundamental role in assessing (patho)physiological cell states, conventional gene reporters can follow gene expression but leave scars on the proteins or substantially alter the mature messenger RNA. Multi-time-point measurements of non-coding RNAs are currently impossible without modifying their nucleotide sequence, which can alter their native function, half-life and localization. Thus, we developed the intron-encoded scarless programmable extranuclear cistronic transcript (INSPECT) as a minimally invasive transcriptional reporter embedded within an intron of a gene of interest. Post-transcriptional excision of INSPECT results in the mature endogenous RNA without sequence alterations and an additional engineered transcript that leaves the nucleus by hijacking the nuclear export machinery for subsequent translation into a reporter or effector protein. We showcase its use in monitoring interleukin-2 (IL2) after T cell activation and tracking the transcriptional dynamics of the long non-coding RNA (lncRNA) NEAT1 during CRISPR interference-mediated perturbation. INSPECT is a method for monitoring gene transcription without altering the mature lncRNA or messenger RNA of the target of interest.

Expression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions., Truong et al. developed a cell-based reporter system, EXSISERS, that enables non-invasive quantification of the protein expression levels of exon-specific isoforms via intein-mediated protein splicing.

Background Many long noncoding RNAs (lncRNAs) have been implicated in general and cell type-specific molecular regulation. Here, we asked what underlies the fundamental basis for the seemingly random appearance of nuclear lncRNA condensates in cells, and we sought compounds that can promote the disintegration of lncRNA condensates in vivo. Results As a basis for comparing lncRNAs and cellular properties among different cell types, we screened lncRNAs in human pluripotent stem cells (hPSCs) that were differentiated to an atlas of cell lineages. We found that paraspeckles, which form by aggregation of the lncRNA NEAT1, are scaled by the size of the nucleus, and that small DNA-binding molecules promote the disintegration of paraspeckles and other lncRNA condensates. Furthermore, we found that paraspeckles regulate the differentiation of hPSCs. Conclusions Positive correlation between the size of the nucleus and the number of paraspeckles exist in numerous types of human cells. The tethering and structure of paraspeckles, as well as other lncRNAs, to the genome can be disrupted by small molecules that intercalate in DNA. The structure-function relationship of lncRNAs that regulates stem cell differentiation is likely to be determined by the dynamics of nucleus size and binding site accessibility. Graphical abstract