Methods for analyzing lariat rna

Abstract

The present invention relates to compositions and methods useful for analyzing lariat RNA, which plays a role in the regulation of gene expression. A sample of RNA is specifically treated to remove linear mRNA and enrich for lariat RNA. The enriched lariat RNA sample may be analyzed further to identify introns, branch point sequences, alternative splicing patters, and gene transcription levels. The enriched lariat RNA sample may also be exploited as a detection or compound screening tool, as well as other uses.

What is claimed is: 
     
         1 . An isolated debranching enzyme comprising an amino acid sequence having at least 35% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 46-66. 
     
     
         2 . The isolated debranching enzyme of  claim 1 , wherein the sequence identity is selected from the group consisting of about 40, 45, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more. 
     
     
         3 . The isolated debranching enzyme of  claim 1 , wherein the amino acid sequence has at least 75% sequence identity to the metallophosphatase domain of a sequence selected from the group consisting of SEQ ID NO: 46-66. 
     
     
         4 . The isolated debranching enzyme of  claim 3 , wherein the sequence identity is selected from the group consisting of about 40, 45, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more. 
     
     
         5 . A method of enriching an RNA population for lariat RNA comprising:
 a. providing an RNA population; and,   b. contacting the RNA population with a linear RNA degrading enzyme to form a lariat RNA enriched population.   
     
     
         6 . The method of  claim 5  further comprising contacting the RNA population with a debranching enzyme. 
     
     
         7 . The method of  claim 6 , wherein the debranching enzyme comprises an amino acid sequence having at least 35% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 46-66. 
     
     
         8 . The method of  claim 5 , wherein the linear RNA degrading enzyme is selected from the group consisting of 3′ exonuclease, 5′ exonuclease, 5′/3′ exonuclease, or combinations thereof. 
     
     
         9 . A method of analyzing lariat RNA in an RNA sample comprising the steps of:
 a. providing an RNA population;   b. contacting the RNA population with a linear RNA degrading enzyme to form a lariat RNA enriched population; and,   c. creating a cDNA library from the lariat RNA population.   
     
     
         10 . The method of  claim 9 , wherein the linear RNA degrading enzyme is selected from the group consisting of 3′ exonuclease, 5′ exonuclease, 5′/3′ exonuclease, or combinations thereof. 
     
     
         11 . The method of  claim 9  further comprising contacting the lariat RNA enriched population with a debranching enzyme. 
     
     
         12 . The method of  claim 10 , wherein the debranching enzyme comprises an amino acid sequence having at least 35% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 46-66. 
     
     
         13 . The method of  claim 9  further comprising sequencing the cDNA library. 
     
     
         14 . A kit comprising:
 a. a linear RNA degrading enzyme;   b. buffer; and,   c. instructions.   
     
     
         15 . The kit of  claim 14  further comprising a debranching enzyme. 
     
     
         16 . The kit of  claim 14  further comprising a debranching enzyme buffer. 
     
     
         17 . The kit of  claim 15 , wherein the debranching enzyme comprises a amino acid sequence having at least 35% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 46-66. 
     
     
         18 . The kit of  claim 17 , wherein the sequence identity is selected from the group consisting of about 40, 45, 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more. 
     
     
         19 . The kit of  claim 14 , wherein the linear RNA degrading enzyme is selected from the group consisting of 3′ exonuclease, 5′ exonuclease, 5′/3′ exonuclease, or combinations thereof. 
     
     
         20 . The kit of  claim 14  further comprising a 5′ decapping enzyme.