Alignments are usually numbered by some reference coordinate system, often a canonical molecule. For instance, tRNA positions are numbered by reference to the positions of yeast tRNA-Phe.
A line beginning with #=RF preceding the sequences in a block
gives a reference coordinate system. Any non-gap symbol in the
#=RF line indicates that sequence positions in its columns are
numbered. For instance, the #=RF lines for a tRNA alignment
would have 76 non-gap symbols for the canonical numbered columns; they
might be the aligned tRNA-Phe sequence itself, or they might be just
X's.