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Back-splicing is an RNA splicing arrangement in which a downstream splice donor site joins an upstream splice acceptor site. In a common circularization event, the retained segment becomes circular RNA with a new junction joining a later part of the precursor to an earlier part. The donor is conventionally called a $5^{\prime }$ splice site and the acceptor a $3^{\prime }$ splice site; those labels describe splice-boundary roles, not the order of their coordinates along the precursor.

For a precursor with ordered exons $1\to 2\to 3\to 4$, a circle containing exons 2 and 3 has the ordinary internal junction $2\to 3$ and a circularizing junction $3\to 2$. A sequence read spanning $3\mid 2$ thus reports a different adjacency from the reference transcript order. The example specifies a possible connectivity, not a prediction that every such precursor forms this circle.

A 2013 study combined RNA sequencing with and without RNase R treatment. Reads crossing back-splice junctions were enriched among the more resistant RNA population, and complementary tests supported circular products.^[ref] A non-colinear junction read alone can also raise questions about mapping, another RNA arrangement or genomic structure. Agreement between junction evidence and a test of molecular connectivity makes the circular interpretation stronger; it does not by itself establish the circle's biological function.

A splice donor site is the splice site at the $5^{\prime }$ end of an intron in a precursor RNA. It marks the boundary between the upstream exon and the intron. Its name is oriented along the precursor RNA, not along whichever direction a DNA reference sequence happens to be displayed.

In the toy precursor A|intron|B, with letters representing whole exons, the first boundary is the splice donor site and the second is the splice acceptor site. Removing the intron and joining A to B gives the mature splice junction. A change in donor choice can change the end of A even when acceptor choice remains fixed; this is one route to alternative splicing.

Recognition involves sequence and molecular context. The RNA component of U1 snRNP can base-pair with the donor region. A particularly informative experiment changed a donor sequence and then changed U1 to restore complementarity. Rescue of one donor mutation supported direct pairing; weak rescue of another showed that complementarity alone did not guarantee productive RNA splicing.^[ref]

This is a useful causal test: perturbing either partner can disrupt an interaction, while a compensating change in the other partner can distinguish a specific pairing model from a generic loss of function. A candidate sequence bound by U1 snRNP still has to participate in the rest of the spliceosome's reaction.