The appeal of using light as a regulatory input is easy to understand. Unlike drugs or small molecules, light can be applied instantly, confined to a specific region, and tuned in intensity without chemically perturbing the cell. Optogenetic circuits built from photosensitive proteins have transformed neuroscience, allowing researchers to map and manipulate neural activity with millisecond precision. But most of these circuits operate at the level of transcription, controlling whether a gene is read into messenger RNA in the first place. Translation, the step in which ribosomes convert mRNA into protein, offers an even more direct and immediate handle on protein levels, and it is the step that cells themselves regulate most heavily during stress, differentiation, and disease.

In living cells, that translational control is largely exerted by RNA-binding proteins, or RBPs, which latch onto specific sequences in messenger RNAs and either block or boost their translation. During epithelial-mesenchymal transition, for example, the RNA-binding protein RBFOX2 is enabled while ESRP1 is disabled, collectively raising the output of mesenchymal-related mRNAs and suppressing epithelial-related ones. Mimicking this kind of opposing, coordinated regulation with a synthetic device has been the missing piece. The HKUST team, led by Yi Kuang, set out to build a platform in which a single light input would activate one RNA-binding protein while simultaneously preventing the formation of another, allowing two different target mRNAs to be regulated in opposite directions at once.

The core of the design is a clever inversion of a standard optogenetic trick. The researchers began with a split intein, a pair of protein fragments derived from the DnaE intein of the cyanobacterium Nostoc punctiforme that spontaneously stitch themselves together and splice out, fusing whatever proteins are attached to their ends. Normally, light-induced dimerization is used to bring protein fragments together. Here, the team did the opposite: they fused one half of the intein to the blue-light dimerizing protein pMag, and the other half to its partner nMag, but inserted a rigid synthetic alpha-helical spacer of about 39.7 angstroms between the intein fragment and pMag. In the dark, the intein halves find each other and splice normally. Under blue light, pMag and nMag dimerize and physically drag the intein halves apart, holding them beyond the reach of the spacer and blocking splicing altogether.

The team validated the concept in HEK293T cells using fluorescent reporters. When cells expressing the two fusion proteins were kept in the dark, the fluorescent signals swapped localization, confirming that the intein had spliced and conjugated the proteins as intended. Western blot analysis sharpened the picture: dark-incubated cells showed a dominant band at 18.3 kilodaltons, the expected spliced product, accounting for 67.6 percent of the signal, while light-exposed cells showed a dominant band at 36.4 kilodaltons, the unspliced precursor, at 71.4 percent. Removing the spacer abolished the light response entirely, with splicing proceeding under both conditions, proving that the rigid helix was the element converting photodimerization into inhibition. The chosen light intensity of roughly 3.75 milliwatts per square centimeter caused no measurable loss of cell viability.

Crucially, the Light-OFF system proved orthogonal to an existing Light-ON intein system based on the light-oxygen-voltage (LOV) sensing domain, which assembles a different engineered intein pair, NpuM, under the same blue light. When both systems were placed in the same cells, one driving reconstitution of split sfGFP under light and the other driving reconstitution of split mCherry in the dark, the two fluorescent outputs cleanly inverted between conditions. sfGFP signal under light reached levels about 4.5-fold higher than in the dark, while mCherry in the dark was roughly twice the light-condition level. The researchers also showed that the Light-OFF mCherry output could be cycled: switching light and dark every 24 hours over five days made the mCherry signal repeatedly appear and disappear without harming the cells.

With the light-sensing machinery in hand, the team turned to the harder problem of engineering the RNA-binding proteins themselves. They chose two widely used model RBPs, the MS2 bacteriophage coat protein (MCP) and the PP7 coat protein (PCP), which bind distinct RNA aptamers and are workhorses for tracking and manipulating RNA in living cells. When bound to aptamers placed in the 5-prime untranslated region of a reporter mRNA, these proteins suppress translation. The challenge was that split inteins prefer particular amino acids at the splicing junction, and the flexible random-coil regions of MCP and PCP did not naturally offer suitable split sites. The researchers inserted cysteine residues at engineered positions and used AlphaFold 3 to simulate whether each candidate split pair would reconstitute into a structure resembling the original protein, screening candidates by template modeling score and root-mean-square deviation before ever testing them in cells.

The simulations paid off. Split MCP with a cysteine inserted at the 38/39 position and split PCP split at 36/37 proved the most potent, each reconstituting into functional repressors that bound their cognate aptamers and suppressed reporter expression. The team then assembled the full bidirectional photoriboswitch: light induces formation of PCP, which suppresses an EGFP reporter bearing the PP7 aptamer, while darkness allows formation of MCP, which suppresses an iRFP reporter bearing the MS2 aptamer. In cells carrying the full device, the EGFP-to-iRFP ratio shifted by more than 4.3-fold between light and dark conditions, and the platform performed similarly in HeLa cells, with a 6.7-fold shift, demonstrating that the effect is not confined to a single cell line.

Perhaps most strikingly, the platform could be flipped from repression to activation. Many natural RNA-binding proteins enhance translation rather than block it, and the team mimicked this by fusing the VPg translation-promoter motif, derived from a viral protein genome-linked factor, onto the split RBP fragments, while fitting the reporter mRNAs with a translation-deficient cap. Now, light-induced formation of PCP-VPg localized the enhancer onto the EGFP switch and boosted its output, while darkness enabled MCP-VPg to upregulate the iRFP switch. The light-to-dark ratio shift in this configuration exceeded 81.7-fold, a dramatic demonstration that the same architectural principle can be adapted to opposing modes of translational control simply by swapping the functional domain attached to the reconstituted protein.

The authors are candid about the current limitations. Blue light, the input used throughout the study, penetrates tissue poorly and can cause DNA damage, so extending the platform to red or near-infrared sensing pairs such as BphP1/QPAS1 will be essential for any move beyond cultured cells. The system also produces irreversible RBP formation, since intein splicing cannot be undone, although natural protein degradation and dilution during cell growth limit the duration of the effect and allow repeated light-dark cycles of regulation. And the demonstration so far regulates two model reporter mRNAs; extending the approach to multiple physiologically relevant transcripts remains future work. Even so, the design principles on display, using reversible photodimerization to gate irreversible protein assembly and using computational structure prediction to engineer split points where none naturally exist, offer a genuinely expandable foundation. As RNA-based therapeutics and synthetic mRNA circuits mature, tools that can dial protein production up and down with nothing more than light are likely to find eager users, from basic researchers dissecting gene regulation to engineers building cell therapies that respond to optical commands.

Subject of Research: A bidirectional light-controlled riboswitch for regulating translation of synthetic mRNAs in mammalian cells

Article Title: Bidirectional photoriboswitch for translational regulation in mammalian cells

Article References: Hu, Y., Li, C. Y., Fu, L., Sun, Y., Zhang, M., Yau, T. M., Xiong, C., Shi, P., & Kuang, Y. (2026). Bidirectional photoriboswitch for translational regulation in mammalian cells. iScience, 29(10), Article 117671. https://doi.org/10.1016/j.isci.2026.117671

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117671

Keywords: synthetic biology, optogenetics, photoriboswitch, translation regulation, RNA-binding proteins, split intein, mRNA, blue light, AlphaFold 3, mammalian cells, gene expression control, HKUST

Inherited retinal dystrophies are a leading cause of irreversible blindness worldwide, and retinitis pigmentosa, the most common form, affects an estimated 2.5 million people. More than 100 causative genes have been identified, yet between 30 and 50 percent of cases still lack a genetic diagnosis, and the observation of incomplete penetrance hints that factors beyond DNA sequence are at work. Epigenetic mechanisms, which modulate gene expression without altering the genome, have increasingly come into focus in ocular disease. Among these, m6A stands out as the most abundant internal modification in eukaryotic messenger RNA, deposited by writer enzymes, removed by erasers such as FTO and ALKBH5, and interpreted by reader proteins that influence splicing, export, stability, and translation.

METTL16 is a relative newcomer to the m6A field. Unlike the well-known METTL3/METTL14 complex, it recognizes a specific motif, UACAGARAA, within structured RNA contexts, and its validated substrates include MAT2A mRNA, which it regulates to maintain cellular levels of the universal methyl donor S-adenosylmethionine, and U6 snRNA, a small nuclear RNA at the heart of the splicing machinery. METTL16 methylates U6 at position A43 within the ACAGAGA sequence that base-pairs with the 5′ splice site of introns during pre-mRNA splicing. Work in fission yeast, roundworms, plants, and human cells has shown that losing this modification impairs 5′ splice site recognition. Intriguingly, dominant mutations in U6 snRNA have recently been linked to autosomal dominant retinitis pigmentosa, and those variants cluster in regions that contact spliceosome components such as PRPF3, PRPF8, and PRPF31, proteins that are themselves mutated in RP.

To probe METTL16’s role in the retina, the team, led by Jiangbo Ren, Wenjing Liu, and colleagues, generated rod-specific Mettl16 knockout mice by crossing Mettl16-floxed animals with RHO-Cre mice, in which the recombinase is active only in rods. The resulting RKO mice showed efficient excision of the targeted exon and a marked reduction of METTL16 protein in the retina. Single-cell RNA sequencing data from the Human Protein Atlas had already indicated that METTL16 is broadly expressed across retinal cell types, with notable enrichment in rods, making the knockout strategy well suited to testing its function in the cells most vulnerable to retinitis pigmentosa.

The physiological consequences were swift and measurable. Electroretinograms recorded at one month of age revealed that scotopic responses, which reflect rod function, were severely blunted: a-wave and b-wave amplitudes were reduced by roughly 54 and 55 percent, respectively, compared with littermate controls. Cone-mediated photopic responses, by contrast, were largely preserved at this stage, pointing to a rod-first pattern of degeneration. Behavioral assays corroborated the electrophysiology. In a light-dark box test, RKO mice spent only about 39 percent of their time and traveled about 42 percent of their distance in the dark chamber, unlike control mice, which normally prefer darkness. Their optomotor response, a reflexive head movement that tracks rotating visual gratings, dropped to 1.56, well below the values of 2.0 or higher seen in controls.

Under the microscope, the story continued. At postnatal day 20, retinal architecture looked normal, but by day 30 the outer nuclear layer and outer segments of RKO retinas were visibly thinning, and by day 40 the outer nuclear layer had lost roughly 40 percent of its thickness relative to controls. Immunofluorescence showed that key outer segment proteins, including rhodopsin, PRPH2, GRK1, PDE6B, and CNGA1, were markedly reduced in abundance even though their localization within the photoreceptor was not disrupted. Signs of degenerative stress were everywhere: Müller glia became reactive, microglia adopted an amoeboid morphology and invaded the outer nuclear layer, and TUNEL staining detected apoptotic nuclei among the photoreceptors. Cones, meanwhile, showed only delayed involvement, with reduced M-opsin-positive cells appearing by day 40.

To trace the molecular roots of this collapse, the researchers integrated RNA sequencing with quantitative proteomics on one-month-old retinas. Among the genes concordantly downregulated at both the transcript and protein levels, Gene Ontology and KEGG analyses pointed squarely at photoreceptor development, photoreceptor cilia, inner and outer segments, visual perception, and the phototransduction cascade. Five established retinitis pigmentosa genes emerged from this analysis: Tulp1, Cnga1, Pde6g, Crb1, and Prcd. Four of them showed significant differential expression at both levels after correction for multiple testing, and the reductions were confirmed by RT-qPCR and Western blotting. The metabolic arm of the analysis revealed perturbations in the S-adenosylmethionine pathway as well, with Mat2a mRNA, a direct METTL16 substrate, reduced in knockout retinas, and Csad, a key enzyme of taurine biosynthesis, markedly decreased.

The mechanistic core of the paper lies in splicing. Using a single-base elongation and ligation-based qPCR assay called SELECT on purified U6 snRNA, the team confirmed that m6A at position A43 was reduced in knockout retinas. Transcriptome-wide analysis with rMATS then revealed extensive alternative splicing changes, with skipped exons as the predominant event class. Sequence analysis of affected 5′ splice sites showed a telling pattern: sites whose usage decreased in knockout retinas were enriched for adenosine at the +4 position, the very nucleotide engaged by methylated U6, and carried an RAG motif across the U6-interacting positions, whereas sites with increased usage favored stronger U5 snRNA base pairing and non-adenine nucleotides at +4. Crucially, aberrant splicing was already detectable at postnatal day 20, before any overt cell loss. Both Tulp1 and Pde6g, two established retinal dystrophy genes, showed increased exon skipping, and the affected donor sites shared the conserved //GURAG motif previously implicated in METTL16 loss in plants and worms. MeRIP-seq showed no change in m6A enrichment on the Tulp1 and Pde6g transcripts themselves, consistent with a U6-dependent mechanism rather than direct methylation of the mRNAs. The Tulp1 exon 4-skipped transcript is predicted to encode an in-frame deletion of 51 amino acids, and immunostaining revealed disrupted ribbon synapse architecture in knockout retinas, echoing the synaptic defects known from Tulp1 knockout mice.

The study also documented METTL16’s influence beyond the nucleus. MeRIP-seq identified Tor1b and Nlgn2 as candidate direct m6A targets, with reduced methylation in their 3′ untranslated regions and reduced expression in knockout retinas; luciferase reporter assays showed that wild-type METTL16 enhanced reporters bearing the wild-type 3′ UTRs but not motif-mutated versions, and a catalytic-domain-deficient METTL16 mutant failed to do so. In photoreceptor-derived 661W cells, loss of METTL16 accelerated the decay of both transcripts, indicating effects on mRNA stability. Meanwhile, immunoprecipitation coupled with mass spectrometry revealed that retinal METTL16 associates with translation factors including PURA, PABPC1, and EIF3B, and with the splicing factors SF3B1 and SF3B3. SUnSET assays and polysome profiling showed that Mettl16 knockdown reduced global translation efficiency in 661W cells, and polysome analysis confirmed diminished translational efficiency of phototransduction genes such as Cep164, Rpgr, Prph2, and Cnga1, whose mRNA levels were unchanged even as their proteins declined.

The authors are careful to note the limitations: most molecular analyses used bulk retinal tissue, which can blur cell-type-specific effects and mix primary METTL16-dependent events with secondary degenerative changes, and the mechanistic work relied on the 661W cell line because mature rods are difficult to manipulate in vitro. Catalytically dead rescue experiments and ribosome profiling will be needed to determine whether the translational role depends on methyltransferase activity. Even so, the picture that emerges is striking. METTL16, working in both the nucleus and the cytoplasm, appears to safeguard photoreceptors on at least three fronts: by methylating U6 snRNA to preserve splicing fidelity in dystrophy genes, by stabilizing specific m6A-marked transcripts, and by supporting the translation of the proteins that build the light-sensing machinery. Given that mutations in U6 snRNA and multiple spliceosome components cause retinitis pigmentosa in humans, the study suggests that RNA processing defects may be a broader and previously underappreciated theme in photoreceptor disease, one that could eventually inform diagnostic and therapeutic strategies for the many patients whose blindness still has no genetic explanation.

Subject of Research: The role of the m6A methyltransferase METTL16 in photoreceptor survival and retinal degeneration

Article Title: METTL16 maintains photoreceptor integrity via splicing fidelity, mRNA stability, and translational regulation

Article References: Ren, J., Liu, W., Zou, R., Sun, K., Yang, M., Zhu, X., & Yang, Z. (2026). METTL16 maintains photoreceptor integrity via splicing fidelity, mRNA stability, and translational regulation. Journal of Advanced Research. https://doi.org/10.1016/j.jare.2026.09.012

Image Credits: AI Generated

DOI: 10.1016/j.jare.2026.09.012

Keywords: METTL16, m6A, photoreceptors, retinitis pigmentosa, U6 snRNA, alternative splicing, RNA methylation, retinal degeneration, translation regulation, Tulp1, Pde6g, epitranscriptomics