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Stable lariats bearing a snoRNA (slb-snoRNA) in eukaryotic cells: A level of regulation for guide RNAs.

ABSTRACT: Small nucleolar (sno)RNAs guide posttranscriptional modifications essential for the biogenesis and function of their target. The majority of snoRNAs in higher eukaryotes are encoded within introns. They are first released from nascent transcripts in the form of a lariat and rapidly targeted by the debranching enzyme and nuclear exonucleases for linearization and further trimming. In this study, we report that some snoRNAs are encoded within unusually stable intronic RNAs. These intronic sequences can escape the debranching enzyme and accumulate as lariats. Stable lariats bearing a snoRNA, or slb-snoRNA, are associated with snoRNA binding proteins but do not guide posttranscriptional modification. While most slb-snoRNAs accumulate in the nucleus, some can be exported to the cytoplasm. We find that this export competes with snoRNA maturation. Slb-snoRNAs provide a previously unknown layer of regulation to snoRNA and snoRNA binding proteins.

SUBMITTER: Talross GJS

PROVIDER: S-EPMC8609340 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. We demonstrate molecular mechanisms for both constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON → OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF → ON logic). For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to design an orthogonal library of three cgRNAs that respond to different RNA triggers. In E. coli expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe a median conditional response of ≈4-fold for an ON → OFF "terminator switch" mechanism, ≈15-fold for an ON → OFF "splinted switch" mechanism, and ≈3-fold for an OFF → ON "toehold switch" mechanism; the median crosstalk within each cgRNA/trigger library is <2%, ≈2%, and ≈20% for the three mechanisms. To test the portability of cgRNA mechanisms prototyped in bacteria to mammalian cells, as well as to test generalizability to different effector functions, we implemented the terminator switch in HEK 293T cells expressing inducing dCas9 as the protein effector, observing a median ON → OFF conditional response of ≈4-fold with median crosstalk of ≈30% for three orthogonal cgRNA/trigger pairs. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for synthetic biology.

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Stable lariats bearing a snoRNA (slb-snoRNA) in eukaryotic cells: A level of regulation for guide RNAs.

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